Design of a targeted next-generation DNA sequencing panel for pediatric T-cell lymphoblastic lymphoma to unravel biology and optimize treatment.

Low incidence and molecular heterogeneity of pediatric T-cell lymphoblastic lymphoma (T-LBL) require an international, large-scale effort to identify novel clinical biomarkers. The ongoing international clinical trial LBL2018 (NCT04043494) represents an ideal opportunity to implement a common analytic approach. Targeted next-generation sequencing is well-suited for this purpose; however, selection of relevant target genes for T-LBL remains subject of ongoing debates. Our group has recently designed and evaluated a first target panel of 80 candidate genes for T-LBL. The present study aimed at developing a novel optimized gene panel for large-scale application and to promote an international agreement on a common core panel. Small sequence variants obtained from our former study were systematically analyzed and classified with regards to pathogenic relevance, to prioritize candidate genes. Additional genes were curated from literature and online databases for a more comprehensive analysis of relevant functions and signaling pathways. The new target panel TGP-T-LBL entails 84 candidate genes which are key actors in NOTCH, PI3K-AKT, JAK-STAT, RAS signaling, epigenetic regulation, transcription, DNA repair, cell cycle regulation, and ribosomal function. From our former gene panel, 35 out of 80 candidate genes were selected for the novel panel. Forty-six out of 84 genes are currently being analyzed in the ongoing international trial LBL2018. Exploratory analysis of prognostic relevance on mutation-level suggested a potential association of PIK3CA variants c.1624G>A(p.Glu542Lys) and c.1633G>A(p.Glu545Lys) to occurrence of relapse, emphasizing particular relevance of mutation analysis in PI3K-AKT signaling. Our approach promotes comprehensive and clinically relevant mutational profiling of pediatric T-LBL.

A phase II trial to evaluate the combination of pixantrone and obinutuzumab for patients with relapsed aggressive lymphoma: Final results of the prospective, multicentre GOAL trial.

The prognosis of patients with relapsed diffuse large B-cell lymphoma (DLBCL) remains poor with current options. Here we prospectively evaluated the combination of pixantrone with obinutuzumab for up to six cycles for patients with relapsed or refractory DLBCL. Overall response rate (ORR) was the primary end-point. Sixty-eight patients were evaluated, median age was 75 years, median number of prior lines was three (range 1-10), 52 patients (76.5%) were diagnosed with DLBCL and 16 (23.5%) patients had transformed indolent lymphoma or follicular lymphoma (FL) IIIB. ORR was 35.3% for all and 40% for evaluable patients (16.6% complete response), median progression-free survival (PFS) and overall survival (OS) were 2.8 months and 8 months, respectively. Analysis of the cell of origin revealed a superior course for patients with non-GCB (germinal centre B-cell-like) phenotype [median OS not reached (n.r.) vs 5.2 months]. Patients with one prior line had an improved outcome over patients treated in later lines (PFS n.r. vs 2.5 months). Disease progression was the main reason for premature termination. Adverse events were mainly haematologic. The combination treatment revealed no unexpected adverse events. Most relevant non-haematologic toxicity was infection in 28% of patients. In summary, pixantrone-obinutuzumab showed clinical activity with sometimes long-term remission; however, the trial failed to meet its primary end-point.

Recurrent CTNNB1 mutations in craniofacial osteomas.

Osteoma is a benign bone forming tumor predominantly arising on the surface of craniofacial bones. While the vast majority of osteomas develops sporadically, a small subset of cases is associated with Gardner syndrome, a phenotypic variant of familial adenomatous polyposis caused by mutations in the APC gene resulting in aberrant activation of WNT/ß-catenin signaling. In a sequencing analysis on a cohort of sporadic, non-syndromal osteomas, we identified hotspot mutations in the CTNNB1 gene (encoding ß-catenin) in 22 of 36 cases (61.1%), harbouring allelic frequencies ranging from 0.04 to 0.53, with the known S45P variant representing the most frequent alteration. Based on NanoString multiplex expression profiling performed in a subset of cases, CTNNB1-mutated osteomas segregated in a defined "WNT-cluster", substantiating functionality of CTNNB1 mutations which are associated with ß-catenin stabilization. Our findings for the first time convincingly show that osteomas represent genetically-driven neoplasms and provide evidence that aberrant WNT/ß-catenin signaling plays a fundamental role in their pathogenesis, in line with the well-known function of WNT/ß-catenin in osteogenesis. Our study contributes to a better understanding of the molecular pathogenesis underlying osteoma development and establishes a helpful diagnostic molecular marker for morphologically challenging cases.

Dietary conjugated linoleic acid links reduced intestinal inflammation to amelioration of CNS autoimmunity.

A close interaction between gut immune responses and distant organ-specific autoimmunity including the CNS in multiple sclerosis has been established in recent years. This so-called gut-CNS axis can be shaped by dietary factors, either directly or via indirect modulation of the gut microbiome and its metabolites. Here, we report that dietary supplementation with conjugated linoleic acid, a mixture of linoleic acid isomers, ameliorates CNS autoimmunity in a spontaneous mouse model of multiple sclerosis, accompanied by an attenuation of intestinal barrier dysfunction and inflammation as well as an increase in intestinal myeloid-derived suppressor-like cells. Protective effects of dietary supplementation with conjugated linoleic acid were not abrogated upon microbiota eradication, indicating that the microbiome is dispensable for these conjugated linoleic acid-mediated effects. Instead, we observed a range of direct anti-inflammatory effects of conjugated linoleic acid on murine myeloid cells including an enhanced IL10 production and the capacity to suppress T-cell proliferation. Finally, in a human pilot study in patients with multiple sclerosis (n = 15, under first-line disease-modifying treatment), dietary conjugated linoleic acid-supplementation for 6 months significantly enhanced the anti-inflammatory profiles as well as functional signatures of circulating myeloid cells. Together, our results identify conjugated linoleic acid as a potent modulator of the gut-CNS axis by targeting myeloid cells in the intestine, which in turn control encephalitogenic T-cell responses.

NTRK testing: First results of the QuiP-EQA scheme and a comprehensive map of NTRK fusion variants and their diagnostic coverage by targeted RNA-based NGS assays.

Gene fusions involving the three neurotrophic tyrosine receptor kinase genes NTRK1, NTRK2, or NTRK3 were identified as oncogenic drivers in many cancer types. Two small molecule inhibitors have been tested in clinical trials recently and require the detection of a NTRK fusion gene prior to therapeutic application. Fluorescence in situ hybridization (FISH) and targeted next-generation sequencing (tNGS) assays are commonly used for diagnostic profiling of gene fusions. In the presented study we applied an external quality assessment (EQA) scheme in order to investigate the suitability of FISH and RNA-/DNA-based tNGS for detection of NTRK fusions in a multinational and multicentric ring trial. In total 27 participants registered for this study. Nine institutions took part in the FISH-based and 18 in the NGS-based round robin test, the latter additionally subdivided into low-input and high-input NGS methods (regarding nucleic acid input). Regardless of the testing method applied, all participants received tumor sections of 10 formalin-fixed and paraffin-embedded (FFPE) tissue blocks for in situ hybridization or RNA/DNA extraction, and the results were submitted via an online questionnaire. For FISH testing, eight of nine (88.8%) participants, and for NGS-based testing 15 of 18 (83.3%) participants accomplished the round robin test successfully. The overall high success rate demonstrates that FISH- and tNGS-based NTRK testing can be well established in a routine diagnostic setting. Complementing this dataset, we provide an updated in silico analysis on the coverage of more than 150 NTRK fusion variants by several commercially available RNA-based tNGS panels.

Testing NTRK testing: Wet-lab and in silico comparison of RNA-based targeted sequencing assays.

NTRK fusions involving three neurotrophic tyrosine receptor kinase genes NTRK1, NTRK2, and NTRK3 and a variety of fusion partners were identified as oncogenic drivers across many cancer types. Drugs that target the chimeric protein product require the identification of the underlying gene fusion. This advocates the diagnostic use of molecular assays ranging from fluorescence in situ hybridization (FISH) and reverse transcription polymerase chain reaction (RT-PCR)/Sanger approaches to targeted next-generation sequencing (NGS). Immunohistochemistry may be used as a screening tool and adjunct diagnostic assay in this context. Although FISH and RT-PCR/Sanger approaches are widely adopted in routine diagnostics, current experience with targeted RNA-based NGS is limited. Here, we report on the analysis of major assays (TruSight TST170 and TruSight RNA Fusion [Illumina]; Archer FusionPlex Solid Tumor, Archer FusionPlex Lung, and Archer FusionPlex Oncology [Archer]; Oncomine Comprehensive Assay v3 RNA and Oncomine Focus RNA [Thermo Fisher Scientific]) that are commercially available. The data set includes performance results of a multicentric comparative wet-lab study as well as an in silico analysis on the ability to detect the broad range of NTRK fusions reported until now. A test algorithm that reflects assay methodology is provided. This data will support implementation of targeted RNA sequencing in routine diagnostics and inform screening and testing strategies that have been brought forward.

Activating mutations in the MAP-kinase pathway define non-ossifying fibroma of bone.

Non-ossifying fibroma (NOF), which occasionally results in pathologic fracture, is considered the most common benign and self-limiting lesion of the growing skeleton. By DNA sequencing we have identified hotspot KRAS, FGFR1 and NF1 mutations in 48 of 59 patients (81.4%) with NOF, at allele frequencies ranging from 0.04 to 0.61. Our findings define NOF as a genetically driven neoplasm caused in most cases by activated MAP-kinase signalling. Interestingly, this driving force either diminishes over time or at least is not sufficient to prevent autonomous regression and resolution. Beyond its contribution to a better understanding of the molecular pathogenesis of NOF, this study adds another benign lesion to the spectrum of KRAS- and MAP-kinase signalling-driven tumours. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Overexpression and Tyr421-phosphorylation of cortactin is induced by three-dimensional spheroid culturing and contributes to migration and invasion of pancreatic ductal adenocarcinoma (PDAC) cells.

BACKGROUND: The nucleation-promoting factor cortactin is expressed and promotes tumor progression and metastasis in various cancers. However, little is known about the biological role of cortactin in the progression of pancreatic ductal adenocarcinoma (PDAC). METHODS: Cortactin and phosphorylated cortactin (Y421) were investigated immunohistochemically in 66 PDAC tumor specimens. To examine the functional role of cortactin in PDAC, we modulated cortactin expression by establishing two cortactin knockout cell lines (Panc-1 and BxPC-3) with CRISPR/Cas9 technique. Cortactin knockout was verified by immunoblotting and immunofluorescence microscopy and functional effects were determined by cell migration and invasion assays. A proteomic screening approach was performed to elucidate potential binding partners of cortactin. RESULTS: Immunohistochemically, we observed higher cortactin expression and Tyr421-phosphorylation in PDAC metastases compared to primary tumor tissues. In PDAC cell lines Panc-1 and BxPC-3, knockdown of cortactin impaired migration and invasion, while cell proliferation was not affected. Three-dimensional spheroid culturing as a model for collective cell migration enhanced cortactin expression and Tyr421-phosphorylation. The activation of cortactin as well as the migratory capacity of PDAC cells could significantly be reduced by dasatinib, a Src family kinase inhibitor. Finally, we identified gelsolin as a novel protein interaction partner of cortactin in PDAC. CONCLUSION: Our data provides evidence that cohesive cell migration induces cortactin expression and phosphorylation as a prerequisite for the gain of an invasive, pro-migratory phenotype in PDAC that can effectively be targeted with dasatinib.

Array-based DNA-methylation profiling in sarcomas with small blue round cell histology provides valuable diagnostic information.

Undifferentiated solid tumors with small blue round cell histology and expression of CD99 mostly resemble Ewing sarcoma. However, they also may include other tumors such as mesenchymal chondrosarcoma, synovial sarcoma, or small cell osteosarcoma. Definitive classification usually requires detection of entity-specific mutations. While this approach identifies the majority of Ewing sarcomas, a subset of lesions remains unclassified and, therefore, has been termed "Ewing-like sarcomas" or small blue round cell tumors not otherwise specified. We developed an approach for further characterization of small blue round cell tumors not otherwise specified using an array-based DNA-methylation profiling approach. Data were analyzed by unsupervised clustering and t-distributed stochastic neighbor embedding analysis and compared with a reference methylation data set of 460 well-characterized prototypical sarcomas encompassing 18 subtypes. Verification was performed by additional FISH analyses, RNA sequencing from formalin-fixed paraffin-embedded material or immunohistochemical marker analyses. In a cohort of more than 1,000 tumors assumed to represent Ewing sarcomas, 30 failed to exhibit the typical EWS translocation. These tumors were subjected to methylation profiling and could be assigned to Ewing sarcoma in 14 (47%), to small blue round cell tumors with CIC alteration in 6 (20%), to small blue round cell tumors with BCOR alteration in 4 (13%), to synovial sarcoma and to malignant rhabdoid tumor in 2 cases each. One single case each was allotted to mesenchymal chondrosarcoma and adamantinoma. 12/14 tumors classified as Ewing sarcoma could be verified by demonstrating either a canonical EWS translocation evading initial testing, by identifying rare breakpoints or fusion partners. The methylation-based assignment of the remaining small blue round cell tumors not otherwise specified also could be verified by entity-specific molecular alterations in 13/16 cases. In conclusion, array-based DNA-methylation analysis of undifferentiated tumors with small blue round cell histology is a powerful tool for precisely classifying this diagnostically challenging tumor group.

SRC inhibition represents a potential therapeutic strategy in liposarcoma.

Liposarcomas (LS) are the most common malignant mesenchymal tumors, with an overall long-term mortality rate of 60%. LS comprise three major subtypes, i.e., well-differentiated/dedifferentiated liposarcoma (WDLS/DDLS), myxoid/round cell liposarcoma (MLS) and pleomorphic liposarcoma (PLS). Aiming at the preclinical identification of novel therapeutic options, we here investigate the functional significance of SRC in primary human LS and in LS-derived cell lines. Immunohistochemical and Western blot analyses reveal relevant levels of activated p-(Tyr416)-SRC in LS of the different subtypes with particular activation in MLS and PLS. Dysregulation of the SRC modifiers CSK and PTP1B was excluded as major reason for the activation of the kinase. Consistent siRNA-mediated knockdown of SRC or inhibition by the SRC inhibitor Dasatinib led to decreased proliferation of LS cell lines of the different subtypes, with MLS cells reacting particularly sensitive in MTT assays. Flow cytometric analyses revealed that this effect was due to a significant decrease in mitotic activity and an induction of apoptosis. SRC inhibition by Dasatinib resulted in dephosphorylation of SRC itself, its interacting partners FAK and IGF-IR as well as its downstream target AKT. Consistent with a particular role of SRC in cell motility, Dasatinib reduced the migratory and invasive potential of MLS cells in Boyden chamber and Matrigel chamber assays. In summary, we provide evidence that SRC activation plays an important role in LS biology and therefore represents a potential therapeutic target, particularly in MLS and PLS.

[Gastrointestinal stromal tumors : Where do we stand?] / Gastrointestinale Stromatumoren : Wo stehen wir?

For more than 20 years gastrointestinal stromal tumors (GIST) have been a paradigm for a targeted treatment with tyrosine kinase inhibitors. A fundamental prerequisite for a neoadjuvant or adjuvant treatment of localized GIST or an additive treatment of metastatic GIST is the molecular typing of tumors, ideally at the initial diagnosis. In addition, the possibility of a hereditary or syndromic predisposition must be considered because this results in consequences for the treatment and a different follow-up strategy.

A Custom Panel for Profiling Microglia Gene Expression.

Despite being immune cells of the central nervous system (CNS), microglia contribute to CNS development, maturation, and homeostasis, and microglia dysfunction has been implicated in several neurological disorders. Recent advancements in single-cell studies have uncovered unique microglia-specific gene expression. However, there is a need for a simple yet elegant multiplexed approach to quantifying microglia gene expression. To address this, we have designed a NanoString nCounter technology-based murine microglia-specific custom codeset comprising 178 genes. We analyzed RNA extracted from ex vivo adult mouse microglia, primary mouse microglia, the BV2 microglia cell line, and mouse bone marrow monocytes using our custom panel. Our findings reveal a pattern where homeostatic genes exhibit heightened expression in adult microglia, followed by primary cells, and are absent in BV2 cells, while reactive markers are elevated in primary microglia and BV2 cells. Analysis of publicly available data sets for the genes present in the panel revealed that the panel could reliably reflect the changes in microglia gene expression in response to various factors. These findings highlight that the microglia panel used offers a swift and cost-effective means to assess microglial cells and can be used to study them in varying contexts, ranging from normal homeostasis to disease models.

Outcome of First-Line Treatment With Pembrolizumab According to KRAS/TP53 Mutational Status for Nonsquamous Programmed Death-Ligand 1-High (≥50%) NSCLC in the German National Network Genomic Medicine Lung Cancer.

INTRODUCTION: Programmed death-ligand 1 expression currently represents the only validated predictive biomarker for immune checkpoint inhibition in metastatic NSCLC in the clinical routine, but it has limited value in distinguishing responses. Assessment of KRAS and TP53 mutations (mut) as surrogate for an immunosupportive tumor microenvironment (TME) might help to close this gap. METHODS: A total of 696 consecutive patients with programmed death-ligand 1-high (≥50%), nonsquamous NSCLC, having received molecular testing within the German National Network Genomic Medicine Lung Cancer between 2017 and 2020, with Eastern Cooperative Oncology Group performance status less than or equal to 1 and pembrolizumab as first-line palliative treatment, were included into this retrospective cohort analysis. Treatment efficacy and outcome according to KRAS/TP53 status were correlated with TME composition and gene expression analysis of The Cancer Genome Atlas lung adenocarcinoma cohort. RESULTS: Proportion of KRASmut and TP53mut was 53% (G12C 25%, non-G12C 28%) and 51%, respectively. In KRASmut patients, TP53 comutations increased response rates (G12C: 69.7% versus 46.5% [TP53mut versus wild-type (wt)], p = 0.004; non-G12C: 55.4% versus 39.5%, p = 0.03), progression-free survival (G12C: hazard ratio [HR] = 0.59, p = 0.009, non-G12C: HR = 0.7, p = 0.047), and overall survival (G12C: HR = 0.72, p = 0.16, non-G12C: HR = 0.56, p = 0.002), whereas no differences were observed in KRASwt patients. After a median follow-up of 41 months, G12C/TP53mut patients experienced the longest progression-free survival and overall survival (33.7 and 65.3 mo), which correlated with high tumor-infiltrating lymphocyte densities in the TME and up-regulation of interferon gamma target genes. Proinflammatory pathways according to TP53 status (mut versus wt) were less enhanced and not different in non-G12C and KRASwt, respectively. CONCLUSIONS: G12C/TP53 comutations identify a subset of patients with a very favorable long-term survival with immune checkpoint inhibitor monotherapy, mediated by highly active interferon gamma signaling in a proinflammatory TME.

NKX3.1 immunohistochemistry and methylome profiling in mesenchymal chondrosarcoma: additional diagnostic value for a well-defined disease?

Mesenchymal chondrosarcoma (MCS) is a rare and highly aggressive tumour of soft tissue and bone that is defined by an underlying and highly specific fusion transcript involving HEY1 and NCOA2. Histologically, the tumours show a biphasic appearance consisting of an undifferentiated blue and round cell component as well as islands of highly differentiated cartilage. Particularly in core needle biopsies, the chondromatous component can be missed and the non-specific morphology and immunophenotype of the round cell component can cause diagnostic challenges. We applied NKX3.1 immunohistochemistry which was recently reported as a highly specific marker as well as methylome and copy number profiling to a set of 45 well characterised MCS cases to evaluate their potential diagnostic value. Methylome profiling revealed a highly distinct cluster for MCS. Notably, the findings were reproducible also when analysing the round cell and cartilaginous component separately. Furthermore, four outliers were identified by methylome profiling for which the diagnosis had to be revised. NKX3.1 immunohistochemistry showed positivity in 36% of tumours, the majority of which was rather focal and weak. Taken together, NKX3.1 expression showed a low sensitivity but a high specificity in our analysis. Methylome profiling on the other hand represents a sensitive, specific and reliable tool to support the diagnosis of MCS, particularly if only the round cell component is obtained in a biopsy and the diagnosis is not suspected. Furthermore, it can aid in confirming the diagnosis in case RNA sequencing for the HEY1::NCOA2 fusion transcript is not available.

Interdependence of SS18-SSX-driven YAP1 and ß-Catenin Activation in Synovial Sarcoma.

Synovial sarcoma, a rare malignant soft tissue tumor, is characterized by a specific chromosomal translocation t(X;18). The resulting chimeric SS18-SSX fusion protein drives synovial sarcoma pathogenesis by integrating into the BAF complex and dysregulating gene transcription. Because previous functional analyses revealed a connection between SS18-SSX and the activity of the transcriptional coregulators YAP1/TAZ and ß-catenin, respectively, this study examined a potential interdependence between these essential effector proteins in synovial sarcoma. In a large cohort of synovial sarcoma tissue specimens, IHC analyses revealed a substantial subset of synovial sarcoma with concurrent nuclear accumulation of YAP1/TAZ and ß-catenin. In vitro, small-molecule inhibitor treatment, RNAi-mediated knockdown, and vector-based overexpression assays demonstrated that YAP1, TAZ, and ß-catenin transcriptional activity is not only stimulated by the SS18-SSX fusion protein, but that they also mutually enhance each other's activation. These analyses showed the highest cooperative effect with overexpression of YAP1 in combination with ß-catenin. Coimmunoprecipitation experiments detected nuclear interactions between YAP1, ß-catenin, and the SS18-SSX fusion protein, the latter being an integral part of the BAF complex. Disruption of BAF complex assembly affected the coregulation of YAP1 and ß-catenin, indicating that this chromatin remodeling complex plays a crucial role for interdependent YAP1 and ß-catenin activation in synovial sarcoma cells. IMPLICATIONS: This study provides deeper insights into synovial sarcoma tumor biology demonstrating a mutual dependence between YAP1/TAZ and ß-catenin transcriptional activity and a complex interplay with the SS18-SSX fusion protein within the BAF complex.

Immunohistochemical expression of TROP­2 (TACSTD2) on the urothelial carcinoma of the urinary bladder and other types of cancer.

In metastatic or locally advanced urothelial carcinoma (UC), therapeutic options have been limited to chemotherapy and immune checkpoint inhibitors. Novel targets and drugs such as antibody drug conjugates have been developed, and enfortumab vedotin targeting nectin-4 and sacituzumab govitecan (SG) targeting trophoblast cell surface antigen 2 (TROP-2), the protein product of the TACSTD2 gene, have been approved. The expression of TROP-2 was investigated within UC and other types of carcinomas, and within the tissue of different healthy organs to understand treatment responses and toxicities. The expression of TROP-2 in the tissues of 42 patients with UC, 13 patients with other types of cancer and in the normal tissues of 11 patients was retrospectively analyzed. Immunohistochemical staining of the TROP-2 protein was performed on a BenchMark ULTRA IHC/ISH System (Roche Tissue Diagnostics; Roche Diagnostics, Ltd.) according to accredited staining protocols in a routine immunohistochemistry accredited and certified facility of the laboratory of immunohistochemistry at the Institute of Pathology (Gerhard-Domagk Institute)- University Hospital Muenster (UKM)-Muenster-Germany]. Different expression levels of TROP-2 were observed, and the highest expression rate of TROP-2 was observed in UC, independent of the tumor stage. However, normal urothelial cells had similar expression levels. Except for ductal carcinoma in situ, the expression of TROP-2 was reduced in other types of cancer and in the healthy tissues from other organs, including pancreas, gall bladder, colon and prostate. Given the treatment response based on the expression level of TROP-2, SG would be effective in almost all cases of UC. However, it would also have an effect on the normal urothelium.

Recurrent EWSR1::COLCA2 Fusions Define a Novel Sarcoma With Spindle/Round Cell Morphology and Strong Predilection for the Sinonasal Tract.

The last 2 decades have attended a dynamic evolution in the nosology of poorly differentiated sinonasal tract malignancies, with several new molecularly defined entities having been described in addition to delineation of the genetic driver/s of some established older entities. These discoveries, however, mostly concerned epithelial-derived neoplasms (carcinomas). Adamantinoma-like Ewing sarcoma and biphenotypic sinonasal sarcoma are the major representatives of the newly defined mesenchymal categories. The colorectal cancer associated 2 (COLCA2) has been discovered recently as a colorectal cancer risk gene locus, but fusions involving this gene have not been well characterized. We, herein, describe clinicopathologic and molecular features of a novel sinonasal sarcoma characterized by undifferentiated spindle/round cell morphology and defined by recurrent EWSR1::COLCA2 fusions. All patients (n=5) were adults (3 female and 2 male) with a median age of 46 years (range, 23 to 60 y). The tumors originated in different subsites of the sinonasal tract with frequent multisite involvement. Original diagnoses were undifferentiated or unclassified round cell/spindle cell neoplasm/sarcoma (n=4) and neuroendocrine carcinoma (n=1). Surgery with or without adjuvant chemoradiation was the treatment in all cases. At the last follow-up, 1 patient developed multiple local recurrences over 21 years and another developed local recurrence and distant metastasis to bone 27 months after diagnosis. A third patient developed local recurrence 11 months later. Two patients were disease-free at 23, and 24 months. Histology showed nondescript highly cellular neoplasms with an admixture of spindled and round cells disposed into solid sheets and fascicles with brisk mitotic activity. Immunohistochemistry was negative for all lineage-specific markers with only limited focal membranous CD99 (4 of 5 cases) and weak pankeratin (1 of 5 cases) expression. Targeted RNA sequencing revealed an EWSR1::COLCA2 fusion, verified by EWSR1 fluorescence in situ hybridization, in all cases. This series identifies a novel member in the undifferentiated spindle/round cell sarcoma category with strong predilection for the sinonasal tract. None of >10,000 epithelial and mesenchymal neoplasms tested at the authors' centers during the same period showed this fusion, highlighting rarity of tumors carrying this gene fusion. Accordingly, molecular testing of unclassified sinonasal malignancies/sarcomas showing round and spindle cell morphology is recommended to enhance the identification and further characterization of this entity.

Molecular profiling of EBV associated diffuse large B-cell lymphoma.

Epstein-Barr virus (EBV) associated diffuse large B-cell lymphoma (DLBCL) represents a rare aggressive B-cell lymphoma subtype characterized by an adverse clinical outcome. EBV infection of lymphoma cells has been associated with different lymphoma subtypes while the precise role of EBV in lymphomagenesis and specific molecular characteristics of these lymphomas remain elusive. To further unravel the biology of EBV associated DLBCL, we present a comprehensive molecular analysis of overall 60 primary EBV positive (EBV+) DLBCLs using targeted sequencing of cancer candidate genes (CCGs) and genome-wide determination of recurrent somatic copy number alterations (SCNAs) in 46 cases, respectively. Applying the LymphGen classifier 2.0, we found that less than 20% of primary EBV + DLBCLs correspond to one of the established molecular DLBCL subtypes underscoring the unique biology of this entity. We have identified recurrent mutations activating the oncogenic JAK-STAT and NOTCH pathways as well as frequent amplifications of 9p24.1 contributing to immune escape by PD-L1 overexpression. Our findings enable further functional preclinical and clinical studies exploring the therapeutic potential of targeting these aberrations in patients with EBV + DLBCL to improve outcome.

Inhibition of p38 signaling curtails the SARS-CoV-2 induced inflammatory response but retains the IFN-dependent antiviral defense of the lung epithelial barrier.

SARS-CoV-2 is the causative agent of the immune response-driven disease COVID-19 for which new antiviral and anti-inflammatory treatments are urgently needed to reduce recovery time, risk of death and long COVID development. Here, we demonstrate that the immunoregulatory kinase p38 MAPK is activated during viral entry, mediated by the viral spike protein, and drives the harmful virus-induced inflammatory responses. Using primary human lung explants and lung epithelial organoids, we demonstrate that targeting p38 signal transduction with the selective and clinically pre-evaluated inhibitors PH-797804 and VX-702 markedly reduced the expression of the pro-inflammatory cytokines IL6, CXCL8, CXCL10 and TNF-α during infection, while viral replication and the interferon-mediated antiviral response of the lung epithelial barrier were largely maintained. Furthermore, our results reveal a high level of drug synergism of both p38 inhibitors in co-treatments with the nucleoside analogs Remdesivir and Molnupiravir to suppress viral replication of the SARS-CoV-2 variants of concern, revealing an exciting and novel mode of synergistic action of p38 inhibition. These results open new avenues for the improvement of the current treatment strategies for COVID-19.

[Abdominal soft tissue tumors]. / Abdominelle Weichgewebstumoren.

Gastrointestinal stromal tumors are the most common mesenchymal tumors in the abdomen and occur in Germany with an incidence of 10 to 15 cases per million inhabitants. Clear identification and characterization are of major importance for the prognosis and therapy of patients. Similarly, they have to be differentiated from other mesenchymal neoplasias such as leiomyomatous, neurogenic, adipocytic, and fibroblastic tumors. Additionally, the number of translocation positive neoplasias is increasing, requiring the use of adequate molecular assays. The aim of this paper is to give practical advice for their identification. Reference pathology is one possibility to support the correct diagnosis.