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PurposeImmune modulatory therapies including immune checkpoint inhibitors have so far failed to result in clinically meaningful efficacy in glioma. We aimed to investigate lymphocyte activation gene 3 (LAG-3), an inhibitory receptor on immune cells and target of second-generation immune checkpoint inhibitors, in glioma.Methods97 patients with diffuse glioma (68 with glioblastoma, 29 with WHO grade II-III glioma) were identified from the Neuro-Biobank of the Medical University of Vienna. LAG-3 expression in the inflammatory microenvironment was assessed by immunohistochemistry (monoclonal anti-LAG-3 antibody, clone 17B4) and correlated to CD3+ , CD8+ , CD20+ and PD-1+ tumor-infiltrating lymphocytes (TILs) and PD-L1 expression on tumor cells.ResultsLAG-3+ TILs could be observed in 10/97 (10.3%) IDH-wildtype samples and in none of the included IDH-mutant glioma samples (p = 0.057). Further, LAG-3+ TILs were only observed in WHO grade IV glioblastoma, while none of the investigated WHO grade II–III glioma presented with LAG-3+ TILs (p = 0.03). No association of O6-methylguanine-DNA-methyltransferase (MGMT) promoter methylation and presence of LAG-3+ TILs was observed (p = 0.726). LAG-3 expression was associated with the presence of CD3+ (p = 0.029), CD8+ (p = 0.001), PD-1+ (p < 0.001) TILs and PD-L1+ tumor cells (p = 0.021), respectively. No association of overall survival with LAG-3+ TIL infiltration was evident (median OS 9.9 vs. 14.2 months, p = 0.95).ConclusionsLAG-3 is only rarely expressed on TILs in IDH-wildtype glioma and associated with active inflammatory milieu as defined by higher TIL density. Immune microenvironment diversity should be considered in the design of future immunotherapy trials in glioma.

High-grade serous ovarian cancer (HGSOC) is an aggressive malignancy which is often treated with platinum-based chemotherapy and PARP inhibitors (PARPi). However, PARPi resistance remains a major clinical challenge, necessitating alternative therapeutic strategies. In this study, we establish the first known patient-derived organoid models directly from PARPi-resistant HGSOC and demonstrate that they preserve the original tumor architecture and key biomarkers (EpCAM, CA125, PAX8, HER2, MEK1/2, Cyclin E1), thus providing unique preclinical models for drug testing. These organoids were used to evaluate lurbinectedin in comparison with standard carboplatin and paclitaxel. While lurbinectedin showed comparable apoptotic effects to paclitaxel and superior activity to carboplatin, it induced chromosomal breaks to different extents in different cell lines, suggesting a distinct mechanism of action. Importantly, this work does not advocate for lurbinectedin as a superior therapy but, rather, demonstrates the utility of organoid models in uncovering drug-specific genomic effects. Our findings underscore the critical need for expanded testing using clinically relevant models to identify more effective strategies against PARPi-resistant disease.

The analytically sensitive detection of D816V in blood and bone marrow is important for diagnosing systemic mastocytosis (SM). Additionally, precise quantification of the D816V variant allele fraction (VAF) is relevant clinically because it helps to predict multilineage involvement and prognosis in cases of advanced SM. Digital PCR (dPCR) is a promising new method for sensitive detection and accurate quantification of somatic mutations. We performed a validation study of dPCR for D816V on 302 peripheral blood and bone marrow samples from 156 patients with mastocytosis for comparison with melting curve analysis after peptide nucleic acid-mediated PCR clamping (clamp-PCR) and allele-specific quantitative real-time PCR (qPCR). dPCR showed a limit of detection of 0.01% VAF with a mean CV of 8.5% and identified the mutation in 90% of patients compared with 70% for clamp-PCR ( < 0.001). Moreover, dPCR for D816V was highly concordant with qPCR without systematic deviation of results, and confirmed the clinical value of D816V VAF measurements. Thus, patients with advanced SM showed a significantly higher D816V VAF (median, 2.43%) compared with patients with indolent SM (median, 0.14%; < 0.001). Moreover, dPCR confirmed the prognostic significance of a high D816V VAF regarding survival ( < 0.001). dPCR for D816V provides a high degree of precision and sensitivity combined with the potential for interlaboratory standardization, which is crucial for the implementation of D816V allele burden measurement. Thus, dPCR is suitable as a new method for D816V testing in patients with mastocytosis.

Oncogenic gene fusions occur across a broad range of cancers and are a defining feature of some cancer types. Cancers driven by gene fusion products tend to respond well to targeted therapies, where available; thus, detection of potentially targetable oncogenic fusions is necessary to select optimal treatment. Detection methods include non-sequencing methods, such as fluorescence in situ hybridization and immunohistochemistry, and sequencing methods, such as DNA- and RNA-based next-generation sequencing (NGS). While NGS is an efficient way to analyze multiple genes of interest at once, economic and technical factors may preclude its use in routine care globally, despite several guideline recommendations. The aim of this review is to present a summary of oncogenic gene fusions, with a focus on fusions that affect tyrosine kinase signaling, and to highlight the importance of testing for oncogenic fusions. We present an overview of the identification of oncogenic gene fusions and therapies approved for the treatment of cancers harboring gene fusions, and summarize data regarding treating fusion-positive cancers with no current targeted therapies and clinical studies of fusion-positive cancers. Although treatment options may be limited for patients with rare alterations, healthcare professionals should identify patients most likely to benefit from oncogenic gene fusion testing and initiate the appropriate targeted therapy to achieve optimal treatment outcomes.

Thymoma and thymic carcinoma are thymic epithelial tumors (TETs). We performed a molecular profiling to investigate the pathogenesis of TETs and identify novel targets for therapy. We analyzed 37 thymomas (18 type A, 19 type B3) and 35 thymic carcinomas. The sequencing of 50 genes detected nonsynonymous mutations in 16 carcinomas affecting ALK, ATM, CDKN2A, ERBB4, FGFR3, KIT, NRAS and TP53. Only two B3 thymomas had a mutation in noncoding regions of the SMARCB1 and STK11 gene respectively. Three type A thymomas harbored a nonsynonymous HRAS mutation. Fluorescence in situ hybridization detected in 38 % of carcinomas a CDKN2A, in 32 % a TP53 and in 8 % an ATM gene deletion, whereas only one B3 thymoma exhibited a CDKNA deletion, and none of the type A thymomas showed a gene loss. Sequencing of the total miRNA pool of 5 type A thymomas and 5 thymic carcinomas identified the C19MC miRNA cluster as highly expressed in type A thymomas, but completely silenced in thymic carcinomas. Furthermore, the miRNA cluster C14MC was downregulated in thymic carcinomas. Among non-clustered miRNAs, the upregulation of miR-21, miR-9-3 and miR-375 and the downregulation of miR-34b, miR-34c, miR-130a and miR-195 in thymic carcinomas were most significant. The expression of ALK, HER2, HER3, MET, phospho-mTOR, p16 INK4A , PDGFRA, PDGFRB, PD-L1, PTEN and ROS1 was investigated by immunohistochemistry. PDGFRA was increased in thymic carcinomas and PD-L1 in B3 thymomas and thymic carcinomas. In summary, our results reveal genetic differences between thymomas and thymic carcinomas and suggest potential novel targets for therapy.

Systemic mastocytosis either presents as aggressive neoplasm with short survival time or indolent systemic mastocytosis with normal life expectancy. In both instances, neoplastic mast cells usually harbor the D816V-mutated variant of KIT. Phenotypically, mast cells in systemic mastocytosis usually express CD25. However, no robust marker that discriminates between aggressive and indolent variants of systemic mastocytosis has been identified yet. We here report that CD30, also known as Ki-1 antigen, is expressed in neoplastic mast cells in a majority of patients with advanced systemic mastocytosis (11/13, 85%), whereas in most patients with indolent systemic mastocytosis (12/45, 27%; P <0.001), only a few if any mast cells stained positive for CD30. These results could be confirmed by TissueFAXS analysis in subsets of patients with indolent systemic mastocytosis ( n =7) and advanced systemic mastocytosis ( n =4; P =0.008). The mast cell leukemia cell line HMC-1, derived from a patient with aggressive systemic mastocytosis also expressed the CD30 protein. In addition, we were able to detect CD30 mRNA in HMC-1 cells as well as in bone marrow biopsy samples in patients with systemic mastocytosis. In contrast, CD30 transcripts could not be detected in bone marrow biopsies in cases of reactive mast cell hyperplasia and in various other myeloid neoplasms. In conclusion, CD30 is preferentially expressed in neoplastic mast cells in advanced mast cell neoplasms. Upregulated expression of CD30 in advanced systemic mastocytosis may thus be employed as a potential marker for grading systemic mastocytosis in hematopathology.

Background Pleural mesothelioma (PM) is an aggressive malignancy with poor prognosis . Unlike many other cancers, PM is mostly characterized by inactivation of tumor suppressor genes. Its highly malignant nature in absence of tumor driving oncogene mutations indicates an extrinsic supply of stimulating signals by cells of the tumor microenvironment (TME). Cancer-associated fibroblasts (CAFs) are an abundant cell type of the TME and have been shown to drive the progression of several malignancies. The aim of the current study was to isolate and characterize patient-derived mesothelioma-associated fibroblasts (Meso-CAFs), and evaluate their impact on PM cells. Methods Meso-CAFs were isolated from surgical specimens of PM patients and analyzed by array comparative genomic hybridization, next generation sequencing, transcriptomics and proteomics. Human PM cell lines were retrovirally transduced with GFP. The impact of Meso-CAFs on tumor cell growth, migration, as well as the response to small molecule inhibitors, cisplatin and pemetrexed treatment was investigated in 2D and 3D co-culture models by videomicroscopy and automated image analysis. Results Meso-CAFs show a normal diploid genotype without gene copy number aberrations typical for PM cells. They express CAF markers and lack PM marker expression. Their proteome and secretome profiles clearly differ from normal lung fibroblasts with particularly strong differences in actively secreted proteins. The presence of Meso-CAFs in co-culture resulted in significantly increased proliferation and migration of PM cells. A similar effect on PM cell growth and migration was induced by Meso-CAF-conditioned medium. Inhibition of c-Met with crizotinib, PI3K with LY-2940002 or WNT signaling with WNT-C59 significantly impaired the Meso-CAF-mediated growth stimulation of PM cells in co-culture at concentrations not affecting the PM cells alone. Meso-CAFs did not provide protection of PM cells against cisplatin but showed significant protection against the EGFR inhibitor erlotinib. Conclusions Our study provides the first characterization of human patient-derived Meso-CAFs and demonstrates a strong impact of Meso-CAFs on PM cell growth and migration, two key characteristics of PM aggressiveness, indicating a major role of Meso-CAFs in driving PM progression. Moreover, we identify signaling pathways required for Meso-CAF-mediated growth stimulation. These data could be relevant for novel therapeutic strategies against PM.

Background: Metastatic pancreatic ductal adenocarcinoma (mPDAC) bears a dismal prognosis due to the limited activity of systemic chemotherapy. In our platform for precision medicine, we aim to offer molecular-guided treatments to patients without further standard therapy options. Methods: In this single center, real-world retrospective analysis of our platform, we describe the molecular-based therapy approaches used in all 50 patients diagnosed with therapy-refractory mPDAC. A molecular portrait of the tumor specimens was created by next-generation sequencing, immunohistochemistry (IHC), microsatellite instability (MSI) testing, and fluorescence in situ hybridization. Results: In total, we detected 123 mutations in 50 patients. The five most frequent mutations were KRAS (n = 40; 80%), TP53 (n = 29; 58%), CDKN2A (n = 8; 16%), SMAD4 (n = 4; 8%), and NOTCH1 (n = 4; 8%), which together accounted for 40.2% of all mutations. Two patients had gene fusions, namely, TBL1XR1–PIK3CA and EIF3E–RSPO2. IHC detected expression of EGFR, phosphorylated mTOR, and PTEN in 36 (72%), 33 (66%), and 17 patients (34%), respectively. For 14 (28%) of the 50 patients, a targeted therapy was suggested based on the identified molecular targets. The recommended treatments included the mTOR inhibitor everolimus (n = 3), pembrolizumab (n = 3), palbociclib (n = 2), nintedanib (n = 2), and cetuximab, crizotinib, tamoxifen, and the combination of lapatinib and trastuzumab, in one patient each. Finally, five patients received the recommended therapy. Four patients died due to disease progression before radiological assessment. One patient was treated with nintedanib and achieved stable disease for 6 months. Conclusion: Based on our observations, precision medicine approaches are feasible and implementable in clinical routine and may provide molecular-based therapy recommendations for mPDAC.

Transforming growth factor beta (TGF‐β) exhibits complex and context‐dependent cellular responses. While it mostly induces tumor‐suppressive effects in early stages of tumorigenesis, tumor‐promoting properties are evident in advanced disease. This TGF‐β duality is still not fully understood, and whether TGF‐β supports invasion and metastasis by influencing cancer cells directly, or rather through the stromal tumor compartment, remains a matter of debate. Here, we utilized a library of colorectal cancer (CRC) patient‐derived tumoroids (PDTs), representing a spectrum of tumor stages, to study cancer cell‐specific responses to TGF‐β. Using conditions allowing for the differentiation of PDTs, we observed TGF‐β‐induced tumor‐suppressive effects in early‐stage tumoroids, whereas more advanced tumoroids were less sensitive to the treatment. Notably, one tumoroid line harboring an atypical KRASQ22K mutation underwent partial epithelial‐to‐mesenchymal transition (EMT), which was associated with morphological changes and increased invasiveness. On a molecular level, this was accompanied by elevated expression of mesenchymal genes, as well as deregulation of pathways associated with matrix remodeling and cell adhesion. Our results suggest that tumor cell‐intrinsic responses to TGF‐β are critical in determining its tumor‐suppressive or tumor‐promoting effects. TGF‐β has a complex role in cancer, exhibiting both tumor‐suppressive and tumor‐promoting properties. Using a series of differentiated tumoroids, derived from different stages and mutational background of colorectal cancer patients, we replicate this duality of TGF‐β in vitro. Notably, the atypical but highly aggressive KRASQ22K mutation rendered early‐stage tumoroids susceptible to TGF‐β treatment and induced partial EMT and increased invasion.

Background: Targeted therapies offer novel opportunities to explore biomarkers based on their mode of action. Taking this into consideration, we evaluated six angiogenesis-related proteins as potential predictive biomarkers, which expression might predict the benefit of bevacizumab treatment in patients with metastatic colorectal cancer (mCRC). Methods: This was a phase II multicenter, two-armed, randomized study, in which patients with mCRC were treated with XELIRI (capecitabine and irinotecan) plus bevacizumab followed by XELOX (capecitabine and oxaliplatin) plus bevacizumab (Arm A) or the reverse sequence (Arm B). Tissue expression level of six prespecified candidates [microvessel density assessed by CD31, PTEN, αV integrin, CD98hc, uPAR and NRP-1] was analyzed via immunohistochemistry. The prognostic impact on survival was quantified using the Cox regression model. The predictive potential for benefit from Arm A versus Arm B treatment was investigated by fitting an interaction between the biomarkers and treatment assignment within a multivariable Cox model. Results: In total, 74 out of 126 patients were included in the analysis. The expression of PTEN, αV integrin, uPAR and NRP-1 was not associated with progression-free survival (PFS) or overall survival (OS). For the first time, we identified that patients with tumors expressing CD98hc had a longer PFS than patients without CD98hc-expression (p = 0.032). More importantly, and in accordance with previous studies, low microvessel density was found to be associated with a reduced PFS [adjusted HR per doubling of CD31-expression (p = 0.53, 95% confidence interval: 0.30–0.95, p = 0.034)]. Conclusions: These results can contribute to the development of a personalized strategy for the treatment of mCRC with bevacizumab.