Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 46
Filter
1.
Life (Basel) ; 12(3)2022 Mar 07.
Article in English | MEDLINE | ID: mdl-35330136

ABSTRACT

In this review, we discuss the development pipeline for transcriptional biomarkers in molecular diagnostics and stress the importance of a reliable gene transcript quantification strategy. Hence, a further focus is put on the MIQE guidelines and how to adapt them for biomarker discovery, from signature validation up to routine diagnostic applications. First, the advantages and pitfalls of the holistic RNA sequencing for biomarker development will be described to establish a candidate biomarker signature. Sequentially, the RT-qPCR confirmation process will be discussed to validate the discovered biomarker signature. Examples for the successful application of RT-qPCR as a fast and reproducible quantification method in routinemolecular diagnostics are provided. Based on the MIQE guidelines, the importance of "key steps" in RT-qPCR is accurately described, e.g., reverse transcription, proper reference gene selection and, finally, the application of automated RT-qPCR data analysis software. In conclusion, RT-qPCR proves to be a valuable tool in the establishment of a disease-specific transcriptional biomarker signature and will have a great future in molecular diagnostics or personalized medicine.

2.
Wien Klin Wochenschr ; 131(19-20): 485-492, 2019 Oct.
Article in English | MEDLINE | ID: mdl-31591676

ABSTRACT

BACKGROUND: Fumaric acid esters are recommended in European guidelines for induction and maintenance treatment of patients with moderate to severe plaque psoriasis. A systemic medication with pure dimethyl fumarate without monoethyl fumarate salts was recently licensed in Europe. OBJECTIVE: The efficacy and safety of pure dimethyl fumarate were assessed in patients with severe (physician global assessment) plaque psoriasis in Austria in the BRIDGE trial. METHODS: In this double blind, randomized, placebo-controlled trial patients received 16-week treatment with pure dimethyl fumarate in a head to head comparison with dimethyl fumarate with monoethyl fumarate salts, which is licensed in Germany. In this post hoc analysis the efficacy and safety were assessed in patients with severe psoriasis in Austria. RESULTS: Efficacy measures significantly improved in both active treatment arms compared to placebo in 65 patients after 16 weeks of treatment. Physician global assessment of clear/almost clear in the dimethyl fumarate group was non-inferior to the dimethyl fumarate with monoethyl fumarate salts group 2 months after end of treatment. No serious adverse reaction occurred in patients with dimethyl fumarate in contrast to the second active treatment. Efficacy outcome was paralleled by quality of life improvements. CONCLUSION: This is the first report of dimethyl fumarate in a severely affected population with plaque psoriasis. Dimethyl fumarate is effective and safe in the systemic treatment of adults with severe psoriasis (physician global assessment).


Subject(s)
Dimethyl Fumarate , Psoriasis , Adult , Aged , Austria , Dimethyl Fumarate/adverse effects , Dimethyl Fumarate/therapeutic use , Double-Blind Method , Europe , Female , Germany , Humans , Male , Middle Aged , Psoriasis/drug therapy , Quality of Life , Severity of Illness Index , Treatment Outcome , Young Adult
3.
EMBO J ; 38(19): e101688, 2019 10 01.
Article in English | MEDLINE | ID: mdl-31475747

ABSTRACT

Lymphatic vessels are essential for skin fluid homeostasis and immune cell trafficking. Whether the lymphatic vasculature is associated with hair follicle regeneration is, however, unknown. Here, using steady and live imaging approaches in mouse skin, we show that lymphatic vessels distribute to the anterior permanent region of individual hair follicles, starting from development through all cycle stages and interconnecting neighboring follicles at the bulge level, in a stem cell-dependent manner. Lymphatic vessels further connect hair follicles in triads and dynamically flow across the skin. At the onset of the physiological stem cell activation, or upon pharmacological or genetic induction of hair follicle growth, lymphatic vessels transiently expand their caliber suggesting an increased tissue drainage capacity. Interestingly, the physiological caliber increase is associated with a distinct gene expression correlated with lymphatic vessel reorganization. Using mouse genetics, we show that lymphatic vessel depletion blocks hair follicle growth. Our findings point toward the lymphatic vasculature being important for hair follicle development, cycling, and organization, and define lymphatic vessels as stem cell niche components, coordinating connections at tissue-level, thus provide insight into their functional contribution to skin regeneration.


Subject(s)
Hair Follicle/growth & development , Lymphatic Vessels/metabolism , Regeneration , Skin Physiological Phenomena , Animals , Cell Cycle , Mice , Stem Cell Niche
4.
J Invest Dermatol ; 139(1): 186-194, 2019 01.
Article in English | MEDLINE | ID: mdl-30009831

ABSTRACT

Chemokines mold the tumor microenvironment by recruiting distinct immune cell populations, thereby strongly influencing disease progression. Previously, we showed that CXCL5 expression is upregulated in advanced stages of primary melanomas, which correlates with the presence of neutrophils in the tumor. The analysis of neutrophil populations in various tissues revealed a distinct phenotype of tumor-associated neutrophils. Tumor-associated neutrophils expressed PD-L1, CXCR4, CCR5, Adam17, and Nos2 and were immunosuppressive in a T-cell proliferation assay. To investigate the impact of CXCL5 and neutrophils in vivo, we established a syngeneic mouse tumor transplantation model using CXCL5-overexpressing and control melanoma cell lines. Growth behavior or vascularization of primary tumors was not affected by CXCL5 expression and neutrophils alone. However, in combination with Poly(I:C), tumor-associated neutrophils were able to attenuate induced antitumoral T-cell responses. CXCL5-overexpressing tumors had reduced lung metastasis compared with control tumors. Neutrophil depletion reversed this effect. In vitro, unstimulated lung-derived neutrophils had higher levels of reactive oxygen species compared with tumor-associated neutrophils, and CXCL5 stimulation further increased reactive oxygen species levels. In summary, in melanoma, neutrophils play a context-dependent role that is influenced by local or systemic factors, and interfere with therapies activating the acquired immune system. Actively switching neutrophils into antitumorigenic mode might be a new therapeutic strategy.


Subject(s)
Chemokine CXCL5/genetics , DNA, Neoplasm/genetics , Gene Expression Regulation, Neoplastic , Melanoma/genetics , Neutrophil Activation/genetics , Neutrophils/metabolism , Skin Neoplasms/genetics , Skin/pathology , Animals , Cell Line, Tumor , Cell Proliferation , Chemokine CXCL5/biosynthesis , Humans , Melanoma/metabolism , Melanoma/pathology , Mice , Neutrophils/pathology , Polymerase Chain Reaction , Skin/metabolism , Skin Neoplasms/metabolism , Skin Neoplasms/pathology , Melanoma, Cutaneous Malignant
5.
Clin Otolaryngol ; 43(5): 1335-1344, 2018 10.
Article in English | MEDLINE | ID: mdl-29992788

ABSTRACT

BACKGROUND: The purpose of this study was to determine whether the expression of 15-lipoxygenase-1 (ALOX15) in primary tumour specimens predicts lymph node metastasis and subsequently clinical outcome in Merkel cell carcinoma (MCC) patients. METHODS: A retrospective medical chart review of 33 patients was performed between 1994 and 2014. Eleven out of 33 (33%) Patients with primary MCC stages I and II were categorised as group I. Twenty two out of 33 (67%) Patients with regional lymph node metastases and/or distant metastases were defined as group II. All available tumour samples were immunostained for ALOX15, Podoplanin and MCPyV large T-protein antibody. RESULTS: ALOX15 expression was observed in 19/23 (83%) primary tumour samples and in all lymph node metastasis. Primary tumours in patients with stage III and IV disease showed a higher expression rate of ALOX15 compared to patients with early stage disease (11/12 (92%) and 8/11 (73%), respectively). In group I, five patients (45%) were MCPyV positive, whereas in group II, 15 patients (68%) were MCPyV positive. The median lymphatic vessel density in ALOX15 negative group I primary tumour samples was lower compared to the median lymphatic vessel density in ALOX15 positive group I primary tumour probes (2.7 range, 1-4.3 vs 4.7 range, 4.0-7.3). Furthermore, all 17 samples of MCC metastases showed ALOX15 expression with a median lymphatic vessel density (not lymph node metastases) of 5.3 (range 2.0-7.3). CONCLUSION: In the current study, we were able to show ALOX15 expression in the primary MCC sample and the metastasis sample. Based on the findings of the current study, expression rate of ALOX15 in primary MCC and metastases is possibly linked to an increased lymphatic vessel density.


Subject(s)
Arachidonate 15-Lipoxygenase/metabolism , Carcinoma, Merkel Cell/metabolism , Carcinoma, Merkel Cell/secondary , Skin Neoplasms/metabolism , Skin Neoplasms/pathology , Aged , Aged, 80 and over , Biomarkers/metabolism , Carcinoma, Merkel Cell/mortality , Female , Humans , Lymphatic Metastasis , Male , Middle Aged , Neoplasm Staging , Predictive Value of Tests , Retrospective Studies , Skin Neoplasms/mortality , Survival Rate
6.
J Invest Dermatol ; 138(7): 1627-1635, 2018 07.
Article in English | MEDLINE | ID: mdl-29474942

ABSTRACT

Chemokines influence tumor metastasis by targeting tumor, stromal, and hematopoietic cells. Characterizing the chemokine mRNA expression profile of human primary melanoma samples, we found CXCL5 significantly up-regulated in stage T4 primary melanomas when compared to thin melanomas (T1 stage). To characterize the role of CXCL5 in melanoma progression, we established a metastasizing murine xenograft model using CXCL5-overexpressing human melanoma cells. CXCL5 had no effect on melanoma proliferation in vitro and on primary tumor growth in vivo, but CXCL5-overexpressing tumors recruited high amounts of neutrophils and exhibited significantly increased lymphangiogenesis in our severe combined immune-deficient mouse model. Recruited neutrophils were found in close proximity to or within lymphatic vessels, often in direct contact with melanoma cells. Clinically, CXCL5-overexpressing melanomas had significantly increased lymph node metastases. We were able to translate these findings to human patient samples and found a positive correlation between CXCL5 expression, numbers of neutrophils in stage T4 primary melanoma, and the occurrence of subsequent locoregional metastasis.


Subject(s)
Chemokine CXCL5/metabolism , Lymphatic Metastasis/immunology , Melanoma/pathology , Neutrophils/immunology , Skin Neoplasms/pathology , Animals , Biomarkers, Tumor , Cell Communication/immunology , Cell Line, Tumor , Chemokine CXCL5/immunology , Female , Follow-Up Studies , Humans , Lymph Nodes/immunology , Lymph Nodes/pathology , Lymphangiogenesis/immunology , Lymphatic Metastasis/pathology , Melanoma/immunology , Mice , Mice, Hairless , Mice, SCID , Neoplasm Staging , Neutrophils/metabolism , RNA, Messenger/metabolism , Skin Neoplasms/immunology , Specific Pathogen-Free Organisms , Spheroids, Cellular , Up-Regulation
7.
Sci Rep ; 7: 40881, 2017 01 19.
Article in English | MEDLINE | ID: mdl-28102348

ABSTRACT

Excessive accumulation of white adipose tissue (WAT) is a hallmark of obesity. The expansion of WAT in obesity involves proliferation and differentiation of adipose precursors, however, the underlying molecular mechanisms remain unclear. Here, we used an unbiased transcriptomics approach to identify the earliest molecular underpinnings occuring in adipose precursors following a brief HFD in mice. Our analysis identifies Heme Oxygenase-1 (HO-1) as strongly and selectively being upregulated in the adipose precursor fraction of WAT, upon high-fat diet (HFD) feeding. Specific deletion of HO-1 in adipose precursors of Hmox1fl/flPdgfraCre mice enhanced HFD-dependent visceral adipose precursor proliferation and differentiation. Mechanistically, HO-1 reduces HFD-induced AKT2 phosphorylation via ROS thresholding in mitochondria to reduce visceral adipose precursor proliferation. HO-1 influences adipogenesis in a cell-autonomous way by regulating events early in adipogenesis, during the process of mitotic clonal expansion, upstream of Cebpα and PPARγ. Similar effects on human preadipocyte proliferation and differentiation in vitro were observed upon modulation of HO-1 expression. This collectively renders HO-1 as an essential factor linking extrinsic factors (HFD) with inhibition of specific downstream molecular mediators (ROS &AKT2), resulting in diminished adipogenesis that may contribute to hyperplastic adipose tissue expansion.


Subject(s)
Cell Differentiation , Cell Proliferation , Heme Oxygenase-1/metabolism , Obesity/pathology , Proto-Oncogene Proteins c-akt/metabolism , 3T3-L1 Cells , Adipocytes/cytology , Adipocytes/metabolism , Adipose Tissue, White/metabolism , Animals , CCAAT-Enhancer-Binding Proteins/metabolism , Diet, High-Fat , Heme Oxygenase-1/antagonists & inhibitors , Heme Oxygenase-1/genetics , Humans , Male , Mice , Mice, Inbred C57BL , Mitochondria/metabolism , PPAR gamma/metabolism , RNA Interference , RNA, Small Interfering/metabolism , Reactive Oxygen Species/metabolism
8.
J Invest Dermatol ; 136(5): 967-977, 2016 05.
Article in English | MEDLINE | ID: mdl-26829032

ABSTRACT

In primary melanoma, the amount of vascular endothelial growth factor C (VEGF-C) expression and lymphangiogenesis predicts the probability of metastasis to sentinel nodes, but conditions boosting VEGF-C expression in melanoma are poorly characterized. By comparative mRNA expression analysis of a set of 22 human melanoma cell lines, we found a striking negative correlation between VEGF-C and microphthalmia-associated transcription factor (MITF) expression, which was confirmed by data mining in GEO databases of human melanoma Affymetrix arrays. Moreover, in human patients, high VEGF-C and low MITF levels in primary melanoma significantly correlated with the chance of metastasis. Pathway analysis disclosed the respective c-Jun N-terminal kinase and p38/mitogen-activated protein kinase activities as being responsible for the inverse regulation of VEGF-C and MITF. Predominant c-Jun N-terminal kinase signaling results in a VEGF-C(low)/MITF(high) phenotype; these melanoma cells are highly proliferative, show low mobility, and are poorly lymphangiogenic. Predominant p38 signaling results in a VEGF-C(high)/MITF(low) phenotype, corresponding to a slowly cycling, highly mobile, lymphangiogenic, and metastatic melanoma. In conclusion, the relative c-Jun N-terminal kinase and p38 activities determine the biological behavior of melanoma. VEGF-C and MITF levels serve as surrogate markers for the respective c-Jun N-terminal kinase and p38 activities and may be used to predict the risk of metastasis in primary melanoma.


Subject(s)
JNK Mitogen-Activated Protein Kinases/metabolism , Lymphangiogenesis/physiology , Melanoma/pathology , Skin Neoplasms/pathology , Vascular Endothelial Growth Factor C/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , Analysis of Variance , Animals , Biomarkers, Tumor/metabolism , Blotting, Western , Cell Proliferation , Disease Models, Animal , Enzyme-Linked Immunosorbent Assay , Female , Humans , Male , Melanoma/metabolism , Mice , Mice, Hairless , Microarray Analysis , Real-Time Polymerase Chain Reaction/methods , Signal Transduction , Skin Neoplasms/metabolism , Statistics, Nonparametric , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
9.
Sex Transm Dis ; 42(4): 223-5, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25763666

ABSTRACT

We present a 47 year old female white HIV-1 infected patient with multiple painless rupioid skin lesions, a CD4 count of 155 cells/mm3, positive syphilis serology and a histopathology conspicuous for malignant syphilis. She could be successfully treated with Benzathine-Benzylpenicillin (Retarpen®) 2,4 Mega I.E., 3x intramuscularly in weekly intervals.


Subject(s)
Anti-Bacterial Agents/administration & dosage , HIV Infections/immunology , HIV-1/immunology , Penicillin G Benzathine/administration & dosage , Syphilis, Cutaneous/drug therapy , Blotting, Western , CD4 Lymphocyte Count , Drug Administration Schedule , Female , HIV Infections/complications , Humans , Middle Aged , Syphilis, Cutaneous/etiology , Syphilis, Cutaneous/immunology , Treatment Outcome , Viral Load
10.
J Invest Dermatol ; 135(1): 212-221, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25084052

ABSTRACT

Melanoma spreads primarily to the sentinel lymph nodes, and its risk correlates with lymphangiogenesis, which is mainly driven by vascular endothelial growth factor (VEGF)-C. However, anti-lymphangiogenic factors are poorly characterized. We have shown in a melanoma model that Wnt1 reduces lymphangiogenesis by reducing VEGF-C expression. Screening this model for additional potentially anti-lymphangiogenic factors identified increased activin A expression and reduced expression of the antagonist, follistatin (FST), in Wnt1(+) cells. Activin A is known to reduce blood vessel formation, but the effects on lymphangiogenesis are unknown. Here we show that human primary melanoma expresses significantly higher levels of activin A and lower levels of FST compared with nevi and melanoma metastasis. Using our mouse model with melanoma cells overexpressing Wnt1, FST, Wnt1/FST, or the inhibin ßA subunit (INHBA, resulting in activin A expression), we found both activin A and Wnt1 to reduce lymphangiogenesis. Whereas Wnt1 also reduced metastasis, this was not seen with activin A. In vitro, activin A phosphorylated SMAD2 in both melanoma and lymphatic endothelium but, although it reduced sprouting of lymphatic endothelium, it enhanced the migration of melanoma cells. In conclusion, activin A is an anti-lymphangiogenic factor, but because of its pleiotropic effects on cell mobility it appears not suitable as a pharmacological target.


Subject(s)
Inhibin-beta Subunits/metabolism , Lymphangiogenesis/physiology , Melanoma/secondary , Skin Neoplasms/pathology , Animals , Cell Line, Tumor , Cell Movement , Disease Models, Animal , Female , Follistatin/metabolism , Humans , Lymph Nodes/metabolism , Lymph Nodes/pathology , Lymphatic Metastasis , Lymphatic Vessels/metabolism , Lymphatic Vessels/pathology , Mice, SCID , Neoplasm Transplantation , Wnt1 Protein/metabolism
11.
Wien Klin Wochenschr ; 125(17-18): 501-7, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23928935

ABSTRACT

BACKGROUND: Merkel cell carcinomas (MCC) are very aggressive tumors of the sun-exposed skin with a high potential to metastasize. Little is known about the genesis of MCC and very few prognostic markers have been detected so far. The Wnt pathway protein ß-catenin and the cell cycle protein cyclin D1 are two promotors of tumor growth and are expressed in a variety of malignant neoplasms such as lymphomas, thyroid, breast cancer, and many others. PATIENTS AND METHODS: Tissue samples of 27 patients with MCC were immunohistochemically stained for ß-catenin and cyclin D1 and correlated with overall survival of patients. In addition, western blot analysis was carried out in the two MCC cell lines MCC-13 and MCC-26. RESULTS: ß-catenin showed a cytoplasmatic expression of 10-30 % in 11 samples and an expression lower than 10 % in eight samples. Nuclear staining was visible in two samples. None of the 27 samples expressed cyclin D1. CONCLUSION: Neither cyclin D1 nor ß-catenin was expressed in a statistically significant manner, concluding that the development of MCCs is independent of ß-catenin and cyclin D1 expression and these proteins are not suitable as prognostic markers. We could describe the expression pattern of cyclin D1 for the first time.


Subject(s)
Biomarkers, Tumor/metabolism , Carcinoma, Merkel Cell/diagnosis , Carcinoma, Merkel Cell/metabolism , Cyclin D1/metabolism , Head and Neck Neoplasms/diagnosis , Head and Neck Neoplasms/metabolism , beta Catenin/metabolism , Aged , Aged, 80 and over , Female , Gene Expression Regulation, Neoplastic , Humans , Male , Middle Aged , Reproducibility of Results , Sensitivity and Specificity
12.
Cell Metab ; 18(1): 62-74, 2013 Jul 02.
Article in English | MEDLINE | ID: mdl-23823477

ABSTRACT

Increased visceral fat is associated with a high risk of diabetes and metabolic syndrome and is in part caused by excessive glucocorticoids (GCs). However, the molecular mechanisms remain undefined. We now identify the GC-dependent gene LIM domain only 3 (LMO3) as being selectively upregulated in a depot-specific manner in human obese visceral adipose tissue, localizing primarily in the adipocyte fraction. Visceral LMO3 levels were tightly correlated with expression of 11ß-hydroxysteroid dehydrogenase type-1 (HSD11B1), the enzyme responsible for local activation of GCs. In early human adipose stromal cell differentiation, GCs induced LMO3 via the GC receptor and a positive feedback mechanism involving 11ßHSD1. No such induction was observed in murine adipogenesis. LMO3 overexpression promoted, while silencing of LMO3 suppressed, adipogenesis via regulation of the proadipogenic PPARγ axis. These results establish LMO3 as a regulator of human adipogenesis and could contribute a mechanism resulting in visceral-fat accumulation in obesity due to excess glucocorticoids.


Subject(s)
Adaptor Proteins, Signal Transducing/physiology , Adipogenesis/physiology , Intra-Abdominal Fat/physiology , LIM Domain Proteins/physiology , Obesity/physiopathology , Up-Regulation/physiology , 11-beta-Hydroxysteroid Dehydrogenase Type 1/physiology , Adaptor Proteins, Signal Transducing/genetics , Adipocytes/pathology , Adipogenesis/genetics , Adult , Animals , Case-Control Studies , Cell Differentiation/physiology , Cells, Cultured , Diabetes Mellitus, Experimental/genetics , Diabetes Mellitus, Experimental/pathology , Diabetes Mellitus, Experimental/physiopathology , Disease Models, Animal , Female , Glucocorticoids/physiology , Humans , Intra-Abdominal Fat/pathology , LIM Domain Proteins/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Mice, SCID , Middle Aged , Obesity/pathology , PPAR gamma/physiology , Up-Regulation/genetics
13.
J Invest Dermatol ; 133(1): 230-8, 2013 Jan.
Article in English | MEDLINE | ID: mdl-22951723

ABSTRACT

Alterations in epidermal growth factor (EGF) expression are known to be of prognostic relevance in human melanoma, but EGF-mediated effects on melanoma have not been extensively studied. As lymph node metastasis usually represents the first major step in melanoma progression, we were trying to identify a potential role of primary tumor-derived EGF in the mediation of melanoma lymph node metastases. Stable EGF knockdown (EGFkd) in EGF-high (M24met) and EGF-low (A375) expressing melanoma cells was generated. Only in EGF-high melanoma cells, EGFkd led to a significant reduction of lymph node metastasis and primary tumor lymphangiogenesis in vivo, as well as impairment of tumor cell migration in vitro. Moreover, EGF-induced sprouting of lymphatic but not of blood endothelial cells was abolished using supernatants of M24met EGFkd cells. In addition, M24met EGFkd tumors showed reduced vascular endothelial growth factor-C (VEGF-C) expression levels. Similarly, in human primary melanomas, a direct correlation between EGF/VEGF-C and EGF/Prox-1 expression levels was found. Finally, melanoma patients with lymph node micrometastases undergoing sentinel node biopsy were found to have significantly elevated EGF serum levels as compared with sentinel lymph node-negative patients. Our data indicate that tumor-derived EGF is important in mediating melanoma lymph node metastasis.


Subject(s)
Epidermal Growth Factor/biosynthesis , Lymphangiogenesis/physiology , Melanoma/pathology , Skin Neoplasms/pathology , Vascular Endothelial Growth Factor C/biosynthesis , Animals , Biomarkers, Tumor/biosynthesis , Biomarkers, Tumor/blood , Cell Movement/genetics , Endothelial Cells/metabolism , Endothelial Cells/pathology , Epidermal Growth Factor/blood , Epidermal Growth Factor/genetics , Female , Gene Knockdown Techniques , Homeodomain Proteins/biosynthesis , Homeodomain Proteins/blood , Humans , Lymphangiogenesis/genetics , Lymphatic Metastasis , Melanoma/metabolism , Mice , Mice, SCID , Neoplasm Micrometastasis/pathology , Sentinel Lymph Node Biopsy , Skin Neoplasms/metabolism , Tumor Cells, Cultured , Tumor Suppressor Proteins/biosynthesis , Tumor Suppressor Proteins/blood , Vascular Endothelial Growth Factor C/blood
15.
Methods ; 59(1): 126-31, 2013 Jan.
Article in English | MEDLINE | ID: mdl-23178393

ABSTRACT

The combination of multiple techniques especially those adding complementary information have proven to be beneficial in terms of data consistency. The employment of quantitative PCR (qPCR) prior to next generation sequencing (NGS) methods such as RNA-Seq and mutational analysis presented here does not only enhance data in terms of CNV integration and sample choice, but also allows a faster and more efficient workflow. Correct analysis of libraries prior to sequencing has proven to be a vital step for specific assumption and to some extent for a more parallel testing. By illustrating the combination of qPCR and NGS in oncological examples, the potential of this approach is presented.


Subject(s)
High-Throughput Nucleotide Sequencing/standards , Neoplasms/genetics , Real-Time Polymerase Chain Reaction/standards , DNA Mutational Analysis/standards , Gene Library , Humans , Neoplasms/metabolism , Quality Control , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Analysis, RNA/standards
17.
J Invest Dermatol ; 132(9): 2235-44, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22572818

ABSTRACT

Wnt signals contribute to melanoma progression by boosting their proliferation and survival. Initially, we expected that activated Wnt signaling also improves their proficiency to recruit blood and lymph vessels. To assess this, we added cell culture supernatants (SNs) of Wnt1(+) and Wnt1(-) melanoma to endothelial spheroids. Whereas SNs of Wnt1(-) melanoma cells induced lymphatic sprouts, those of Wnt1(+) cells were unable to do so and this was restored by vascular endothelial growth factor C (VEGF-C). Subsequent testing of several human melanoma lines revealed that Wnt1 suppressed their VEGF-C expression. This Wnt1 effect did not depend on glycogen synthase kinase-3ß (GSK3ß), ß-catenin, or activator protein-1, but was blocked by cyclosporine A (CsA). To analyze Wnt1 effects in melanoma in vivo, we selected Wnt1(-) melanoma cell lines, overexpressed Wnt1, and injected them subepidermally into severe combined immunodeficient (SCID) mice. We found reduced VEGF-C expression, reduced lymphangiogenesis, and delayed metastasis to sentinel nodes in Wnt1(+) as compared with Wnt1(-) melanoma (P<0.05). Concomitant overexpression of VEGF-C or feeding of animals with CsA restored lymphangiogenesis and metastasis in Wnt1(+) melanoma. In conclusion, Wnt1 is anti-lymphangiogenic by suppressing melanoma-derived VEGF-C expression.


Subject(s)
Lymphangiogenesis , Vascular Endothelial Growth Factor C/antagonists & inhibitors , Wnt1 Protein/biosynthesis , Animals , Cyclosporine/pharmacology , Disease Models, Animal , Female , Humans , Lymphatic Metastasis , Melanoma/pathology , Mice , Mice, Inbred C57BL , Mice, SCID , Skin Neoplasms/pathology , Tumor Cells, Cultured , Vascular Endothelial Growth Factor C/biosynthesis , Wnt Signaling Pathway/physiology , Wnt1 Protein/genetics
18.
Head Neck ; 33(9): 1294-300, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21837699

ABSTRACT

BACKGROUND: An overexpression of PIN1, the peptidyl-prolyl cis-trans isomerase, might cause cell cycle arrest and growth inhibition by binding to the p53 protein, a process leading to p53 stabilization. The rationale of this retrospective analysis was to evaluate the expression pattern of PIN1 in Merkel cell carcinomas (MCCs) and its suitability as a prognostic factor. METHODS: Samples of 27 MCCs were immunhistochemically stained for PIN1 expression and correlated with overall and disease-free survival of patients. RESULTS: All samples expressed PIN1. We showed a significantly better overall survival in patients with an overexpression of PIN1 than in patients with a weak PIN1 expression (p = .031), but expression was not significant for disease-free survival (p = .821). The 5-year overall survival rate was 14.4% in patients with weak and 50.9% in patients with overexpression of PIN1. CONCLUSIONS: PIN1 seems to be a prognostic factor for a better overall survival rate of patients with MCC.


Subject(s)
Carcinoma, Merkel Cell/enzymology , Carcinoma, Merkel Cell/mortality , Peptidylprolyl Isomerase/metabolism , Skin Neoplasms/enzymology , Skin Neoplasms/mortality , Aged , Aged, 80 and over , Carcinoma, Merkel Cell/pathology , Female , Humans , Immunohistochemistry , Male , Middle Aged , NIMA-Interacting Peptidylprolyl Isomerase , Prognosis , Retrospective Studies , Skin Neoplasms/pathology , Survival Analysis
19.
Eur Arch Otorhinolaryngol ; 268(11): 1639-46, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21331782

ABSTRACT

The aim of this retrospective analysis was to evaluate the status of p53 and possible mutations in Merkel cell carcinoma (MCC) cell lines and MCC tissue samples. The p53 mutations are common in different cancer origins but rare in MCCs detected so far. MCCs are highly aggressive neuroendocrine tumors with an enhanced potential to metastasize. Until now, less is known about MCC and new approaches to understand this disease are necessary. RNA and DNA were extracted from two MCC cell lines and 27 archival paraffin-embedded patient samples. After reverse transcription, a real-time PCR and a high-resolution melt analysis were carried out. In both MCC cell lines, we could detect a p53 missense mutation at codon 193 (exon 6) with a change in amino acids (His → Leu). This mutation was equal in both cell lines and was investigated in 27 tissue samples in succession to detect possible accounts for the aggressive behavior of MCCs. Unfortunately, no corresponding p53 mutation could be observed in the investigated tissue samples. A new p53 mutation was detected in MCC cell lines. This mutation could not be determined in patients' samples. Therefore, the aggressiveness of MCC seems to be independent of p53 mutations and other mutations might be responsible for developing MCC.


Subject(s)
Carcinoma, Merkel Cell/genetics , DNA, Neoplasm/genetics , Genes, p53/genetics , Head and Neck Neoplasms/genetics , Mutation , Skin Neoplasms/genetics , Aged , Aged, 80 and over , Blotting, Western , Carcinoma, Merkel Cell/pathology , Cell Line, Tumor , Female , Follow-Up Studies , Head and Neck Neoplasms/pathology , Humans , Immunohistochemistry , Male , Middle Aged , Prognosis , Real-Time Polymerase Chain Reaction , Retrospective Studies , Skin Neoplasms/pathology
20.
Magn Reson Med ; 65(6): 1718-23, 2011 Jun.
Article in English | MEDLINE | ID: mdl-21254205

ABSTRACT

MRI can be used to investigate human skin microvasculature in vivo, provided adequate spatial resolution. Therefore, the sensitivity of the experiment has to be optimized to achieve sufficient signal-to-noise ratio (SNR) within reasonable measurement time to minimize motion artifacts, improve patient comfort and save costs. In this work, the high sensitivity of a 15 mm surface coil and the signal strength of a 3 Tesla scanner, together with a three-dimensional gradient echo sequence and post-processing have been combined to obtain high SNR. Images of human skin with isotropic spatial resolution of 100 µm were acquired within 10 min and the cutaneous vasculature could be visualized in 3D [Correction made here after initial online publication.], based on three averaged scans. The presented method can be used for diagnosis and, due to its non-invasiveness, treatment monitoring of vascular pathologies in the skin, such as inflammation, vascular malformation, or neoangiogenesis in superficial tumors.


Subject(s)
Magnetic Resonance Imaging/methods , Skin/blood supply , Vascular Diseases/diagnosis , Adult , Artifacts , Female , Humans , Image Processing, Computer-Assisted , Imaging, Three-Dimensional , Male , Patient Positioning , Reproducibility of Results , Sensitivity and Specificity
SELECTION OF CITATIONS
SEARCH DETAIL
...