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2.
Cell ; 143(2): 201-11, 2010 Oct 15.
Article in English | MEDLINE | ID: mdl-20946980

ABSTRACT

Signaling by ErbB receptors requires the activation of their cytoplasmic kinase domains, which is initiated by ligand binding to the receptor ectodomains. Cytoplasmic factors contributing to the activation are unknown. Here we identify members of the cytohesin protein family as such factors. Cytohesin inhibition decreased ErbB receptor autophosphorylation and signaling, whereas cytohesin overexpression stimulated receptor activation. Monitoring epidermal growth factor receptor (EGFR) conformation by anisotropy microscopy together with cell-free reconstitution of cytohesin-dependent receptor autophosphorylation indicate that cytohesins facilitate conformational rearrangements in the intracellular domains of dimerized receptors. Consistent with cytohesins playing a prominent role in ErbB receptor signaling, we found that cytohesin overexpression correlated with EGF signaling pathway activation in human lung adenocarcinomas. Chemical inhibition of cytohesins resulted in reduced proliferation of EGFR-dependent lung cancer cells in vitro and in vivo. Our results establish cytohesins as cytoplasmic conformational activators of ErbB receptors that are of pathophysiological relevance.


Subject(s)
Adenocarcinoma/pathology , ErbB Receptors/metabolism , Guanine Nucleotide Exchange Factors/metabolism , Lung Neoplasms/pathology , Receptor Protein-Tyrosine Kinases/metabolism , Adenocarcinoma/metabolism , Animals , Dimerization , GTPase-Activating Proteins/antagonists & inhibitors , GTPase-Activating Proteins/genetics , GTPase-Activating Proteins/metabolism , Gene Knockdown Techniques , Guanine Nucleotide Exchange Factors/antagonists & inhibitors , Guanine Nucleotide Exchange Factors/genetics , Humans , Lung Neoplasms/metabolism , Mice , Neoplasm Transplantation , Protein Structure, Tertiary , Signal Transduction , Transplantation, Heterologous , Triazoles/pharmacology
3.
Pathologe ; 42(1): 103-115, 2021 Feb.
Article in German | MEDLINE | ID: mdl-33258061

ABSTRACT

NTRK gene fusions are sporadic genetic alterations that can occur across tumor entities. Whereas they are quite rare in most solid tumors they are present at much higher frequencies in certain rare tumors such as infantile fibrosarcoma, congenital mesoblastic nephroma, secretory breast, or salivary gland carcinoma. NTRK gene fusions or TRK fusion proteins are considered strong oncogenic drivers. If NTRK gene fusions are detected, TRK inhibitors such as entrectinib and larotrectinib can be used regardless of the tumor entity. So far only larotrectinib is approved in the European Union. Both drugs have been shown to be effective and well tolerated in phase I and phase II studies. The low prevalence of TRK fusion-positive cancers poses challenges for diagnostic and clinical work-flows. On one hand, patients with NTRK gene fusions should be identified; on the other hand, epidemiological, histological, and resource-related aspects have to be taken into account. Based on these premises, we suggest a diagnostic algorithm for TRK fusion cancers and present current data on TRK inhibitors.


Subject(s)
Kidney Neoplasms , Nephroma, Mesoblastic , Gene Fusion/genetics , Genetic Markers , Humans , Mutation , Oncogene Proteins, Fusion/genetics , Protein Kinase Inhibitors/therapeutic use , Receptor, trkA/genetics
4.
Int J Cancer ; 146(11): 3053-3064, 2020 06 01.
Article in English | MEDLINE | ID: mdl-31970771

ABSTRACT

Cancer of unknown primary (CUP) denotes a malignancy with histologically confirmed metastatic spread while the primary tumor remains elusive. Here, we address prognostic and therapeutic implications of mutations and copy number variations (CNVs) detected in tumor tissue in the context of a comprehensive clinical risk assessment. Targeted panel sequencing was performed in 252 CUP patients. 71.8% of patients had unfavorable CUP according to ESMO guidelines. 74.7% were adeno- and 13.7% squamous cell carcinomas. DNA was extracted from microdissected formalin-fixed, paraffin-embedded tissues. For library preparation, mostly multiplex PCR-based Ion Torrent AmpliSeq™ technology with Oncomine comprehensive assays was used. Most frequent genetic alterations were mutations/deletions of TP53 (49.6%), CDKN2A (19.0%) and NOTCH1 (14.1%) as well as oncogenic activation of KRAS (23.4%), FGFR4 (14.9%) and PIK3CA (10.7%). KRAS activation was predominantly found in adenocarcinomas (p = 0.01), PIK3CA activation in squamous cell carcinomas (p = 0.03). Male sex, high ECOG score, unfavorable CUP, higher number of involved organs and RAS activation predicted decreased event-free and overall survival in multivariate analysis. Deletions of CDKN2A were prognostically adverse regarding overall survival. TP53 mutations did not significantly influence prognosis in the overall cohort, but worsened prognosis in otherwise favorable CUP subtypes. Although not standard in CUP, for 17/198 (8.6%) patients molecularly targeted treatment was recommended and 10 patients (5.1%) were treated accordingly. In conclusion, besides the identification of drug targets, panel sequencing in CUP is prognostically relevant, with RAS activation and CDKN2A deletion emerging as novel independent risk factors in a comprehensive assessment with clinicopathological data.


Subject(s)
Adenocarcinoma/genetics , Carcinoma, Squamous Cell/genetics , Cyclin-Dependent Kinase Inhibitor p16/genetics , Neoplasms, Unknown Primary/genetics , Proto-Oncogene Proteins p21(ras)/genetics , Adenocarcinoma/pathology , Adolescent , Adult , Carcinoma, Squamous Cell/pathology , Class I Phosphatidylinositol 3-Kinases/genetics , DNA Copy Number Variations/genetics , Female , High-Throughput Nucleotide Sequencing , Humans , Male , Neoplasms, Unknown Primary/pathology , Receptor, Fibroblast Growth Factor, Type 4/genetics , Receptor, Notch1/genetics , Tumor Suppressor Protein p53/genetics , Young Adult
5.
Int J Mol Sci ; 21(7)2020 Apr 05.
Article in English | MEDLINE | ID: mdl-32260486

ABSTRACT

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) regulates target gene expression upon ligand binding. Apart from its effects on metabolism, PPARγ activity can inhibit the production of pro-inflammatory cytokines by several immune cells, including dendritic cells and macrophages. In chronic inflammatory disease models, PPARγ activation delays the onset and ameliorates disease severity. Here, we investigated the effect of PPARγ activation by the agonist Pioglitazone on the function of hepatic immune cells and its effect in a murine model of immune-mediated hepatitis. Cytokine production by both liver sinusoidal endothelial cells (IL-6) and in T cells ex vivo (IFNγ) was decreased in cells from Pioglitazone-treated mice. However, PPARγ activation did not decrease pro-inflammatory tumor necrosis factor alpha TNFα production by Kupffer cells after Toll-like receptor (TLR) stimulation ex vivo. Most interestingly, although PPARγ activation was shown to ameliorate chronic inflammatory diseases, it did not improve hepatic injury in a model of immune-mediated hepatitis. In contrast, Pioglitazone-induced PPARγ activation exacerbated D-galactosamine (GalN)/lipopolysaccharide (LPS) hepatitis associated with an increased production of TNFα by Kupffer cells and increased sensitivity of hepatocytes towards TNFα after in vivo Pioglitazone administration. These results unravel liver-specific effects of Pioglitazone that fail to attenuate liver inflammation but rather exacerbate liver injury in an experimental hepatitis model.


Subject(s)
Hepatitis, Autoimmune/immunology , PPAR gamma/agonists , Pioglitazone/pharmacology , Animals , Cells, Cultured , Interferon-gamma/metabolism , Kupffer Cells/drug effects , Kupffer Cells/immunology , Lymphocyte Activation , Macrophage Activation , Mice , Mice, Inbred C57BL , PPAR gamma/metabolism , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , Toll-Like Receptors/metabolism , Tumor Necrosis Factor-alpha/metabolism
6.
Int J Mol Sci ; 20(22)2019 Nov 06.
Article in English | MEDLINE | ID: mdl-31698731

ABSTRACT

Myeloid cells are essential for the initiation and termination of innate and adaptive immunity that create homeostasis in the liver. Smad7 is an inhibitor of the transforming growth factor ß (TGF-ß) signaling pathway, which regulates inflammatory cellular processes. Knockdown of Smad7 in hepatocytes has been shown to promote liver fibrosis, but little is known about the effects of Smad7 in myeloid cells during inflammatory responses in the liver. Using mice with a myeloid-specific knockdown of Smad7 (LysM-Cre Smad7fl/fl), we investigated the impact of Smad7 deficiency in myeloid cells on liver inflammation and regeneration using the well-established model of CCl4-mediated liver injury. Early (24/48 h) and late (7 d) time points were analyzed. We found that CCl4 induces severe liver injury, with elevated serum ALT levels, centrilobular and periportal necrosis, infiltrating myeloid cells and an increase of inflammatory cytokines in the liver. Furthermore, as expected, inflammation peaked at 24 h and subsided after 7 d. However, the knockdown of Smad7 in myeloid cells did not affect any of the investigated parameters in the CCl4-treated animals. In summary, our results suggest that the inhibition of TGF-ß signaling via Smad7 expression in myeloid cells is dispensable for the induction and control of acute CCl4-induced liver injury.


Subject(s)
Carbon Tetrachloride/administration & dosage , Liver/injuries , Liver/metabolism , Myeloid Cells/metabolism , Acute Disease , Animals , Cell Cycle/genetics , Gene Expression Regulation , Inflammation/genetics , Inflammation/pathology , Liver/pathology , Liver Regeneration , Male , Mice
7.
Histopathology ; 72(3): 449-459, 2018 Feb.
Article in English | MEDLINE | ID: mdl-28851100

ABSTRACT

AIMS: Programmed death ligand 1 (PD-L1) immunohistochemistry has become a mandatory diagnostic test in the treatment of lung cancer. Several research initiatives have started to harmonise the five PD-L1 immunohistochemistry assays that have been used in clinical trials. Here, we report data on interlaboratory and interassay concordance for commercial assays ('assays') and laboratory-developed tests (LDTs) at 10 German testing sites. METHODS AND RESULTS: To assess interlaboratory concordance, a tissue microarray containing 21 pulmonary carcinoma specimens was centrally prepared. Pre-cut sections were stained at 10 sites by the use of assays 28-8, 22C3, SP263, and SP142, as well as 11 LDTs. Assay performance was evaluated with a second tissue microarray containing 11 cell lines with defined PD-L1 expression. Quality control was centrally performed by manual and digital analyses. The assays yielded reproducible IHC staining patterns at all sites. In agreement with previous studies, 22C3, 28-8 and SP263 showed similar staining patterns, whereas SP142 was distinct. Among the LDTs, six of 11 protocols showed staining patterns similar to those of assays 22C3 and 28-8. Interlaboratory concordance of tumour cell scoring by use of a six-step system was moderate (Light's κ = 0.43-0.69), whereas the clinically approved cut-offs of ≥1% and ≥50% showed substantial concordance (κ = 0.73-0.89). Immune cell scoring by the use of SP142 yielded moderate concordance (κ = 0.42). CONCLUSIONS: The data confirm the previously described staining patterns of the assays, and show that they can be reproducibly employed at different sites. LDTs with staining results similar to those of the assays are implementable, but have to be carefully validated.


Subject(s)
B7-H1 Antigen/analysis , Biomarkers, Tumor/analysis , Carcinoma, Non-Small-Cell Lung/diagnosis , Immunohistochemistry/standards , Lung Neoplasms/diagnosis , Humans , Reproducibility of Results
8.
Int J Cancer ; 138(4): 927-38, 2016 Feb 15.
Article in English | MEDLINE | ID: mdl-26340530

ABSTRACT

Small cell lung cancers (SCLCs) and extrapulmonary small cell cancers (SCCs) are very aggressive tumors arising de novo as primary small cell cancer with characteristic genetic lesions in RB1 and TP53. Based on murine models, neuroendocrine stem cells of the terminal bronchioli have been postulated as the cellular origin of primary SCLC. However, both in lung and many other organs, combined small cell/non-small cell tumors and secondary transitions from non-small cell carcinomas upon cancer therapy to neuroendocrine and small cell tumors occur. We define features of "small cell-ness" based on neuroendocrine markers, characteristic RB1 and TP53 mutations and small cell morphology. Furthermore, here we identify a pathway driving the pathogenesis of secondary SCLC involving inactivating NOTCH mutations, activation of the NOTCH target ASCL1 and canonical WNT-signaling in the context of mutual bi-allelic RB1 and TP53 lesions. Additionally, we explored ASCL1 dependent RB inactivation by phosphorylation, which is reversible by CDK5 inhibition. We experimentally verify the NOTCH-ASCL1-RB-p53 signaling axis in vitro and validate its activation by genetic alterations in vivo. We analyzed clinical tumor samples including SCLC, SCC and pulmonary large cell neuroendocrine carcinomas and adenocarcinomas using amplicon-based Next Generation Sequencing, immunohistochemistry and fluorescence in situ hybridization. In conclusion, we identified a novel pathway underlying rare secondary SCLC which may drive small cell carcinomas in organs other than lung, as well.


Subject(s)
Carcinoma, Neuroendocrine/genetics , Carcinoma, Neuroendocrine/pathology , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Signal Transduction , Small Cell Lung Carcinoma/genetics , Small Cell Lung Carcinoma/pathology , Basic Helix-Loop-Helix Transcription Factors/metabolism , DNA Mutational Analysis , Flow Cytometry , Fluorescent Antibody Technique , High-Throughput Nucleotide Sequencing , Humans , Immunohistochemistry , In Situ Hybridization, Fluorescence , Receptors, Notch/genetics , Receptors, Notch/metabolism , Retinoblastoma Protein/genetics , Transfection , Tumor Suppressor Protein p53/genetics
9.
Mod Pathol ; 29(10): 1165-72, 2016 10.
Article in English | MEDLINE | ID: mdl-27389313

ABSTRACT

Immunohistochemistry of the PD-L1 protein may be predictive for anti-PD-1 and anti-PD-L1 immunotherapy in pulmonary adenocarcinoma and in clinically unselected cohorts of so-called non-small-cell lung cancer. Several PD-L1 immunohistochemistry assays with custom reagents and scoring-criteria are developed in parallel. Biomarker testing and clinical decision making would profit from harmonized PD-L1 diagnostics. To assess interobserver concordance and PD-L1 immunohistochemistry staining patterns, 15 pulmonary carcinoma resection specimens (adenocarcinoma: n=11, squamous-cell carcinoma: n=4) were centrally stained with the assays 28-8, 22C3, SP142, and SP263 according to clinical trial protocols. The slides were evaluated independently by nine pathologists. Proportions of PD-L1-positive carcinoma cells and immune cells were scored according to a 6-step system that integrates the criteria employed by the four PD-L1 immunohistochemistry assays. Proportion scoring of PD-L1-positive carcinoma cells showed moderate interobserver concordance coefficients for the 6-step scoring system (Light's kappa=0.47-0.50). The integrated dichotomous proportion cut-offs (≥1, ≥5, ≥10, ≥50%) showed good concordance coefficients (κ=0.6-0.8). Proportion scoring of PD-L1-positive immune cells yielded low interobserver concordance coefficients both for the 6-step-score (κ<0.2) and the dichotomous cut-offs (κ=0.12-0.25). The assays 28-8 and 22C3 stained similar proportions of carcinoma cells in 12 of 15 cases. SP142 stained fewer carcinoma cells compared to 28-8, 22C3, and SP263 in four cases, whereas SP263 stained more carcinoma cells in nine cases. SP142 and SP263 stained immune cells more intensely. The data indicate that carcinoma cells can be reproducibly scored in PD-L1 immunohistochemistry for pulmonary adenocarcinoma and squamous-cell carcinoma. No differences in interobserver concordance were noticed among the tested assays. The scoring of immune cells yielded low concordance rates and might require specific standardization. The four tested PD-L1 assays did not show comparable staining patterns in all cases. Thus, studies that correlate staining patterns and response to immunotherapy are required to test the significance of the observed differences.


Subject(s)
Adenocarcinoma , B7-H1 Antigen/analysis , Biomarkers, Tumor/analysis , Carcinoma, Squamous Cell , Lung Neoplasms , Humans , Immunohistochemistry/methods , Observer Variation
10.
Am J Pathol ; 185(11): 3025-38, 2015 Nov.
Article in English | MEDLINE | ID: mdl-26506472

ABSTRACT

The immunoregulatory cytokine IL-10 suppresses T-cell immunity. The complementary question, whether IL-10 is also involved in limiting the collateral damage of vigorous T cell responses, has not been addressed in detail. Here, we report that the particularly strong virus-specific immune response during acute primary infection with the lymphocytic choriomeningitis virus (LCMV) in mice is significantly further increased in Il10-deficient mice, particularly regarding frequencies and cytotoxic activity of CD8(+) T cells. This increase results in exacerbating immunopathology in select organs, ranging from transient local swelling to an increased risk for mortality. Remarkably, LCMV-induced, T cell-mediated hepatitis is not affected by endogenous Il10. The alleviating effect of Il10 on LCMV-induced immunopathology was found to be operative in delayed-type hypersensitivity footpad-swelling reaction and in debilitating meningitis in mice of both the C57BL/6 and BALB/c strains. These strains are prototypic counterpoles for genetically imprinted type 1-biased versus type 2-biased T cell-mediated immune responses against various infectious pathogens. However, during acute LCMV infection, neither systemic cytokine patterns nor the impact of Il10 on LCMV-induced immunopathology differed conspicuously between these two strains of mice. This study documents a physiological role of Il10 in the regulation of a balanced T-cell response limiting immunopathological damage.


Subject(s)
Antiviral Agents/immunology , CD8-Positive T-Lymphocytes/immunology , Immunity, Cellular , Interleukin-10/immunology , Lymphocytic Choriomeningitis/immunology , Lymphocytic choriomeningitis virus/immunology , Animals , Antiviral Agents/metabolism , CD8-Positive T-Lymphocytes/physiology , Cytokines/blood , Cytokines/immunology , Female , Hypersensitivity, Delayed , Interleukin-10/genetics , Interleukin-10/metabolism , Lymphocytic Choriomeningitis/physiopathology , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout
11.
Exp Mol Pathol ; 99(3): 682-6, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26546837

ABSTRACT

Small cell lung carcinoma (SCLC) is the most aggressive entity of lung cancer. Rapid cancer progression and early formation of systemic metastases drive the deadly outcome of SCLC. Recent advances in identifying oncogenes by cancer whole genome sequencing improved the understanding of SCLC carcinogenesis. However, tumor material is often limited in the clinic. Thus, it is a compulsive issue to improve SCLC diagnostics by combining established immunohistochemistry and next generation sequencing. We implemented amplicon-based next generation deep sequencing in our routine diagnostics pipeline to analyze RB1, TP53, EP300 and CREBBP, frequently mutated in SCLC. Thereby, our pipeline combined routine SCLC histology and identification of somatic mutations. We comprehensively analyzed fifty randomly collected SCLC metastases isolated from trachea and lymph nodes in comparison to specimens derived from primary SCLC. SCLC lymph node metastases showed enhanced proliferation and frequently a collapsed keratin cytoskeleton compared to SCLC metastases isolated from trachea. We identified characteristic synchronous mutations in RB1 and TP53 and non-synchronous CREBBP and EP300 mutations. Our data showed the benefit of implementing deep sequencing into routine diagnostics. We here identify oncogenic drivers and simultaneously gain further insights into SCLC tumor biology.


Subject(s)
DNA Mutational Analysis/methods , High-Throughput Nucleotide Sequencing/methods , Lung Neoplasms/genetics , Neoplasm Metastasis/genetics , Small Cell Lung Carcinoma/genetics , Humans , Lung Neoplasms/pathology , Neoplasm Metastasis/diagnosis , Small Cell Lung Carcinoma/pathology
12.
Proc Natl Acad Sci U S A ; 109(42): 17034-9, 2012 Oct 16.
Article in English | MEDLINE | ID: mdl-23035247

ABSTRACT

Small cell lung cancer (SCLC) accounts for about 15% of all lung cancers. The prognosis of SCLC patients is devastating and no biologically targeted therapeutics are active in this tumor type. To develop a framework for development of specific SCLC-targeted drugs we conducted a combined genomic and pharmacological vulnerability screen in SCLC cell lines. We show that SCLC cell lines capture the genomic landscape of primary SCLC tumors and provide genetic predictors for activity of clinically relevant inhibitors by screening 267 compounds across 44 of these cell lines. We show Aurora kinase inhibitors are effective in SCLC cell lines bearing MYC amplification, which occur in 3-7% of SCLC patients. In MYC-amplified SCLC cells Aurora kinase inhibition associates with G2/M-arrest, inactivation of PI3-kinase (PI3K) signaling, and induction of apoptosis. Aurora dependency in SCLC primarily involved Aurora B, required its kinase activity, and was independent of depletion of cytoplasmic levels of MYC. Our study suggests that a fraction of SCLC patients may benefit from therapeutic inhibition of Aurora B. Thus, thorough chemical and genomic exploration of SCLC cell lines may provide starting points for further development of rational targeted therapeutic intervention in this deadly tumor type.


Subject(s)
Enzyme Inhibitors/pharmacology , G2 Phase Cell Cycle Checkpoints/physiology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Signal Transduction/drug effects , Small Cell Lung Carcinoma/drug therapy , Small Cell Lung Carcinoma/genetics , Apoptosis/drug effects , Aurora Kinase B , Aurora Kinases , Benzothiazoles , Cell Line, Tumor , Cell Survival/drug effects , DNA Primers/genetics , Diamines , Flow Cytometry , G2 Phase Cell Cycle Checkpoints/drug effects , Humans , Immunoblotting , Organic Chemicals , Phosphoinositide-3 Kinase Inhibitors , Proto-Oncogene Proteins c-myc/metabolism , Quinolines , Reverse Transcriptase Polymerase Chain Reaction
13.
Int J Cancer ; 134(12): 2829-40, 2014 Jun 15.
Article in English | MEDLINE | ID: mdl-24242212

ABSTRACT

NKG2D, an activating receptor expressed on NK cells and T cells, is critically involved in tumor immunosurveillance. In this study, we explored the potential therapeutic utility of the NKG2D ligand ULBP2 for the treatment of colon carcinoma. To this end we designed a fusion protein consisting of human ULBP2 and an antibody-derived single chain targeting the tumor carcinoembryonic antigen (CEA). The bispecific recombinant fusion protein re-directed NK cells towards malignant cells by binding to both, tumor cells and NK cells, and triggered NK cell-mediated target cell killing in vitro. Moreover, tumor growth was significantly delayed in a syngeneic colon carcinoma mouse model in response to immunoligand treatment. The anti-tumor activity could be attributed to the stimulation of immune cells with an elevated expression of the activation marker CD69 on NK, T and NKT cells and the infiltration of CD45+ immune cells into the solid tumor. In summary, it was demonstrated that immunoligands provide specific tumor targeting by NK cells and exert anti-tumor activity in vitro and in vivo. This technology represents a novel immunotherapeutic strategy for solid tumors with the potential to be further developed for clinical applications.


Subject(s)
Carcinoembryonic Antigen/immunology , Colonic Neoplasms/immunology , Colonic Neoplasms/therapy , Intercellular Signaling Peptides and Proteins/therapeutic use , NK Cell Lectin-Like Receptor Subfamily K/immunology , Natural Killer T-Cells/immunology , Adoptive Transfer , Animals , Antigens, CD/metabolism , Antigens, Differentiation, T-Lymphocyte/metabolism , Carcinoembryonic Antigen/genetics , Cell Line, Tumor , Disease Models, Animal , GPI-Linked Proteins/therapeutic use , HEK293 Cells , Humans , Immunotherapy, Adoptive , Killer Cells, Natural/immunology , Killer Cells, Natural/transplantation , Lectins, C-Type/metabolism , Leukocyte Common Antigens/metabolism , Mice , Mice, Inbred C57BL , Recombinant Fusion Proteins/therapeutic use , Single-Chain Antibodies/genetics , Single-Chain Antibodies/immunology
14.
Blood ; 120(10): 2042-54, 2012 Sep 06.
Article in English | MEDLINE | ID: mdl-22837530

ABSTRACT

Primary cutaneous lymphomas (PCLs) are clonal T- or B-cell neoplasms, which originate in the skin. In recent years, mast cells were described as regulators of the tumor microenvironment in different human malignancies. Here, we investigated the role of mast cells in the tumor microenvironment of PCL. We found significantly increased numbers of mast cells in skin biopsies from patients with cutaneous T-cell lymphoma (CTCL) and cutaneous B-cell lymphoma (CBCL). Mast cell infiltration was particularly prominent in the periphery, at lymphoma rims. Interestingly, CTCL and CBCL patients with a progressive course showed higher mast cell counts than stable patients, and mast cell numbers in different stages of CTCL correlated positively with disease progression. In addition, mast cell numbers positively correlated with microvessel density. Incubating primary CTCL cells with mast cell supernatant, we observed enhanced proliferation and production of cytokines. In line with our in vitro experiments, in a mouse model of cutaneous lymphoma, tumor growth in mast cell-deficient transgenic mice was significantly decreased. Taken together, these experiments show that mast cells play a protumorigenic role in CTCL and CBCL. Our data provide a rationale for exploiting tumor-associated mast cells as a prognostic marker and therapeutic target in PCL.


Subject(s)
Cell Transformation, Neoplastic/pathology , Lymphocytes/pathology , Lymphoma, T-Cell, Cutaneous/pathology , Mast Cells/pathology , Skin Neoplasms/pathology , Animals , Biomarkers/analysis , Biopsy , Cell Count , Cell Line , Cell Movement , Cell Proliferation/drug effects , Cell Transformation, Neoplastic/immunology , Cell Transformation, Neoplastic/metabolism , Culture Media, Conditioned/pharmacology , Cytokines/biosynthesis , Cytokines/immunology , Humans , Lymphocytes/drug effects , Lymphocytes/metabolism , Lymphoma, T-Cell, Cutaneous/immunology , Lymphoma, T-Cell, Cutaneous/metabolism , Mast Cells/immunology , Mast Cells/metabolism , Mice , Mice, Transgenic , Primary Cell Culture , Prognosis , Skin Neoplasms/immunology , Skin Neoplasms/metabolism , Tumor Microenvironment
15.
Urol Int ; 93(1): 113-8, 2014.
Article in English | MEDLINE | ID: mdl-24556868

ABSTRACT

BACKGROUND: Epigenetic alterations, including histone modifications, play an important role during carcinogenesis. This study was designed to systematically investigate histone H3K9 and H3K27 methylation levels in bladder cancer (BCa) tissue. METHODS: A tissue microarray with urothelial BCa (150 non-muscle-invasive BCa, NMIBC; 121 muscle-invasive BCa, MIBC; 31 metastatic BCa, MET) and normal urothelium (29, CTRL) specimen was used to determine the global levels of H3K9 and H3K27 mono-, di- and tri-methylation. RESULTS: Global levels of H3K9 and H3K27 methylation were significantly higher in CTRL than in BCa, and levels in NMIBC were higher compared to MIBC. Histone methylation levels of MET resembled MIBC. We observed furthermore a correlation of histone methylation levels with pT stage (H3K9me1, H3K9me2, H3K9me3, H3K27me1, H3K27me3) and grade (H3K9me2, H3K9me3, H3K27me1) in NMIBC. H3K9me1, H3K9me3, H3K27me1 and H3K27me3 levels were also correlated with pT stage in MIBC. Histone modifications were not associated with recurrence-free or cancer-specific survival. CONCLUSIONS: Global histone H3K9 and H3K27 methylation levels are altered in BCa.


Subject(s)
Histones/metabolism , Urinary Bladder Neoplasms/metabolism , Adult , Aged , Aged, 80 and over , Female , Humans , Immunohistochemistry , Lymphatic Metastasis , Male , Methylation , Middle Aged , Neoplasm Recurrence, Local/pathology , Prognosis , Tissue Array Analysis , Urothelium/pathology
16.
Transl Lung Cancer Res ; 13(7): 1749-1755, 2024 Jul 30.
Article in English | MEDLINE | ID: mdl-39118880

ABSTRACT

Background: Capmatinib, a potent and selective MET tyrosine kinase inhibitor (TKI), holds promise as a therapeutic agent due to its potentially elevated intracranial efficacy in metastatic non-small cell lung cancer (NSCLC) patients harboring exon 14 skipping alterations in MET (MET Proto-Oncogene). This study aims to evaluate a targeted therapeutic approach to an MET exon 14 skipping (METex14) advanced NSCLC patient that progressed on Crizotinib and developed off target resistance alteration in PIK3CA. Case Discription: We present a case of advanced METex14 NSCLC patient wherein central nervous system (CNS) relapse occurred post complete surgical resection and remission of the lung tumor under first-line crizotinib treatment. Subsequent disease monitoring demonstrated a profound intracranial response to capmatinib in a crizotinib-resistant brain lesion. Molecular analysis unveiled the original METex14 D1028N driver mutation and a newly arisen PIK3CA bypass mutation, potentially contributing to off-target resistance. Conclusions: Before capmatinib was approved as a first line treatment option for metastatic NSCLC harboring somatic METex14 mutations, crizotinib conferred a potential option for targeted treatment. Switching to a selective MET-TKI like capmatinib with a better CNS penetration, it appears to be a promising approach for CNS metastasized NSCLC patients with METex14 mutations that failed on crizotinib. Further research is needed to more effectively understand and monitor resistance mechanisms using advanced diagnostic techniques such as DNA-based hybrid-capture (HC) next generation sequencing (NGS) to guide molecularly stratified therapy beyond the first line setting.

17.
JCO Precis Oncol ; 8: e2300348, 2024 Mar.
Article in English | MEDLINE | ID: mdl-38513168

ABSTRACT

PURPOSE: Poly(ADP-ribose) polymerase inhibitors (PARPi) have shown promising clinical results in the treatment of ovarian cancer. Analysis of biomarker subgroups consistently revealed higher benefits for patients with homologous recombination deficiency (HRD). The test that is most often used for the detection of HRD in clinical studies is the Myriad myChoice assay. However, other assays can also be used to assess biomarkers, which are indicative of HRD, genomic instability (GI), and BRCA1/2 mutation status. Many of these assays have high potential to be broadly applied in clinical routine diagnostics in a time-effective decentralized manner. Here, we compare the performance of a multitude of alternative assays in comparison with Myriad myChoice in high-grade serous ovarian cancer (HGSOC). METHODS: DNA from HGSOC samples was extracted from formalin-fixed paraffin-embedded tissue blocks of cases previously run with the Myriad myChoice assay, and GI was measured by multiple molecular assays (CytoSNP, AmoyDx, Illumina TSO500 HRD, OncoScan, NOGGO GISv1, QIAseq HRD Panel and whole genome sequencing), applying different bioinformatics algorithms. RESULTS: Application of different assays to assess GI, including Myriad myChoice, revealed high concordance of the generated scores ranging from very substantial to nearly perfect fit, depending on the assay and bioinformatics pipelines applied. Interlaboratory comparison of assays also showed high concordance of GI scores. CONCLUSION: Assays for GI assessment not only show a high concordance with each other but also in correlation with Myriad myChoice. Thus, almost all of the assays included here can be used effectively to assess HRD-associated GI in the clinical setting. This is important as PARPi treatment on the basis of these tests is compliant with European Medicines Agency approvals, which are methodologically not test-bound.


Subject(s)
BRCA1 Protein , Ovarian Neoplasms , Humans , Female , BRCA1 Protein/genetics , Mutation , BRCA2 Protein/genetics , Ovarian Neoplasms/diagnosis , Ovarian Neoplasms/genetics , Ovarian Neoplasms/drug therapy , Genomic Instability/genetics , Homologous Recombination/genetics
18.
J Hepatol ; 59(3): 528-35, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23665041

ABSTRACT

BACKGROUND & AIMS: Myeloid derived suppressor cells (MDSCs) are a heterogeneous population of cells associated with the suppression of immunity. However, little is known about how or where MDSCs are induced and from which cells they originate. The liver is known for its immune regulatory functions. Here, we investigated the capacity of human hepatic stellate cells (HSCs) to transform peripheral blood monocytes into MDSCs. METHODS: We cultured freshly isolated human monocytes from healthy donors on primary human HSCs or an HSC cell-line and characterized the phenotype and function of resulting CD14(+)HLA-DR(-/low) monocytes by flow cytometry, quantitative PCR, and functional assays. We analyzed the molecular mechanisms underlying the induction and function of the CD14(+)HLA-DR(-/low) cells by using blocking antibodies or knock-down technology. RESULTS: Mature peripheral blood monocytes co-cultured with HSCs downregulated HLA-DR and developed a phenotypic and functional profile similar to MDSCs. Only activated but not freshly isolated HSCs were capable of inducing CD14(+)HLA-DR(-/low) cells. Such CD14(+)HLA-DR(-/low) monocyte-derived MDSCs suppressed T-cell proliferation in an arginase-1 dependent fashion. HSC-induced development of CD14(+)HLA-DR(-/low) monocyte-derived MDSCs was not mediated by soluble factors, but required physical interaction and was abrogated by blocking CD44. CONCLUSIONS: Our study shows that activated human HSCs convert mature peripheral blood monocytes into MDSCs. As HSCs are activated during chronic inflammation, the subsequent local induction of MDSCs may prevent ensuing excessive liver injury. HSC-induced MDSCs functionally and phenotypically resemble those isolated from liver cancer patients. Thus, our data suggest that local generation of MDSCs by liver-resident HSCs may contribute to immune suppression during inflammation and cancer in the liver.


Subject(s)
Hepatic Stellate Cells/immunology , Hyaluronan Receptors/metabolism , Monocytes/cytology , Monocytes/immunology , Myeloid Cells/cytology , Myeloid Cells/immunology , Arginase/antagonists & inhibitors , Arginase/metabolism , Cell Communication/immunology , Cell Differentiation/immunology , Cell Line , Cell Proliferation/drug effects , Coculture Techniques , Down-Regulation , HLA-DR Antigens/metabolism , Humans , Immune Tolerance , Lipopolysaccharide Receptors/metabolism , Lymphocyte Activation , T-Lymphocytes/cytology , T-Lymphocytes/drug effects , T-Lymphocytes/immunology
19.
Curr Oncol ; 30(10): 8805-8814, 2023 09 27.
Article in English | MEDLINE | ID: mdl-37887535

ABSTRACT

EGFR-mutant lung cancers develop a wide range of potential resistance alterations under therapy with the third-generation EGFR tyrosine kinase inhibitor osimertinib. MET amplification ranks among the most common acquired resistance alterations and is currently being investigated as a therapeutic target in several studies. Nevertheless, targeted therapy of MET might similarly result in acquired resistance by point mutations in MET, which further expands therapeutic and diagnostic challenges. Here, we report a 50-year-old male patient with EGFR-mutant lung adenocarcinoma and stepwise acquired resistance by a focal amplification of MET followed by D1246N (D1228N), D1246H (D1228H), and L1213V (L1195V) point mutations in MET, all detected by NGS. The patient successfully responded to the combined and sequential treatment of osimertinib, osimertinib/crizotinib, and third-line osimertinib/cabozantinib. This case highlights the importance of well-designed, sequential molecular diagnostic analyses and the personalized treatment of patients with acquired resistance.


Subject(s)
Lung Neoplasms , Humans , Male , Middle Aged , Crizotinib/therapeutic use , Drug Resistance, Neoplasm/genetics , ErbB Receptors/genetics , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Mutation , Protein Kinase Inhibitors/adverse effects , Proto-Oncogene Proteins c-met/genetics
20.
Cells ; 13(1)2023 12 30.
Article in English | MEDLINE | ID: mdl-38201288

ABSTRACT

Synaptopodin-2 (SYNPO2) is a protein associated with the Z-disc in striated muscle cells. It interacts with α-actinin and filamin C, playing a role in Z-disc maintenance under stress by chaperone-assisted selective autophagy (CASA). In smooth muscle cells, SYNPO2 is a component of dense bodies. Furthermore, it has been proposed to play a role in tumor cell proliferation and metastasis in many different kinds of cancers. Alternative transcription start sites and alternative splicing predict the expression of six putative SYNPO2 isoforms differing by extended amino- and/or carboxy-termini. Our analyses at mRNA and protein levels revealed differential expression of SYNPO2 isoforms in cardiac, skeletal and smooth muscle cells. We identified synemin, an intermediate filament protein, as a novel binding partner of the PDZ-domain in the amino-terminal extension of the isoforms mainly expressed in cardiac and smooth muscle cells, and demonstrated colocalization of SYNPO2 and synemin in both cell types. A carboxy-terminal extension, mainly expressed in smooth muscle cells, is sufficient for association with dense bodies and interacts with α-actinin. SYNPO2 therefore represents an additional and novel link between intermediate filaments and the Z-discs in cardiomyocytes and dense bodies in smooth muscle cells, respectively. In pathological skeletal muscle samples, we identified SYNPO2 in the central and intermediate zones of target fibers of patients with neurogenic muscular atrophy, and in nemaline bodies. Our findings help to understand distinct functions of individual SYNPO2 isoforms in different muscle tissues, but also in tumor pathology.


Subject(s)
Actinin , Myocytes, Smooth Muscle , Humans , Myocytes, Cardiac , Protein Isoforms , Sarcomeres
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