Publications by authors named "Stephan N Wagner"

45 Publications

Loss of Lymphotoxin Alpha-Expressing Memory B Cells Correlates with Metastasis of Human Primary Melanoma.

Diagnostics (Basel) 2021 Jul 12;11(7). Epub 2021 Jul 12.

Laboratory of Molecular Dermato-Oncology and Tumor Immunology, Department of Dermatology, Medical University of Vienna, 1090 Vienna, Austria.

Activated antigen-experienced B cells play an unexpected complex role in anti-tumor immunity in human melanoma patients. However, correlative studies between B cell infiltration and tumor progression are limited by the lack of distinction between functional B cell subtypes. In this study, we examined a series of 59 primary and metastatic human cutaneous melanoma specimens with B cell infiltration. Using seven-color multiplex immunohistochemistry and automated tissue imaging and analysis, we analyzed the spatiotemporal dynamics of three major antigen-experienced B cell subpopulations expressing lymphotoxin alpha (LTA/TNFSF1) or interleukin-10 (IL-10) outside tertiary lymphoid structures. The expression of both LTA and IL-10 was not restricted to a particular B cell subtype. In primary melanomas, these cells were predominantly found at the invasive tumor-stroma front and, in metastatic melanomas, they were also found in the intratumoral stroma. In primary melanomas, decreased densities of LTA memory-like and, to a lesser extent, activated B cells were associated with metastasis. Compared with metastatic primary tumors, B cell infiltrates in melanoma metastases were enriched in both LTA memory-like and LTA activated B cells, but not in any of the IL-10 B cell subpopulations. Melanoma disease progression shows distinct dynamics of functional B cell subpopulations, with the regulation of LTA B cell numbers being more significant than IL-10 B cell subpopulations.
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http://dx.doi.org/10.3390/diagnostics11071238DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8307480PMC
July 2021

A Standardized Analysis of Tertiary Lymphoid Structures in Human Melanoma: Disease Progression- and Tumor Site-Associated Changes With Germinal Center Alteration.

Front Immunol 2021 24;12:675146. Epub 2021 Jun 24.

Laboratory of Molecular Dermato-Oncology and Tumor Immunology, Department of Dermatology, Medical University of Vienna, Vienna, Austria.

There is increasing evidence that tertiary lymphoid structures (TLS) control not only local adaptive B cell responses at melanoma tumor sites but also the cellular composition and function of other immune cells. In human melanoma, however, a comprehensive analysis of TLS phenotypes, density and spatial distribution at different disease stages is lacking. Here we used 7-color multiplex immunostaining of whole tissue sections from 103 human melanoma samples to characterize TLS phenotypes along the expression of established TLS-defining molecular and cellular components. TLS density and spatial distribution were determined by referring TLS counts to the tissue area within defined intra- and extratumoral perimeters around the invasive tumor front. We show that only a subgroup of primary human melanomas contains TLS. These TLS rarely formed germinal centers and mostly located intratumorally within 1 mm distance to the invasive tumor front. In contrast, melanoma metastases had a significantly increased density of secondary follicular TLS. They appeared preferentially in stromal areas within an extratumoral 1 mm distance to the invasive tumor front and their density varied over time and site of metastasis. Interestingly, secondary follicular TLS in melanoma often lacked BCL6 lymphatic cells and canonical germinal center polarity with the formation of dark and light zone areas. Our work provides an integrated qualitative, quantitative and spatial analysis of TLS in human melanoma and shows disease progression- and site-associated changes in TLS phenotypes, density and spatial distribution. The frequent absence of canonical germinal center polarity in melanoma TLS highlights the induction of TLS maturation as a potential additive to future immunotherapy studies. Given the variable evaluation strategies used in previous TLS studies of human tumors, an important asset of this study is the standardized quantitative evaluation approach that provides a high degree of reproducibility.
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http://dx.doi.org/10.3389/fimmu.2021.675146DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8264652PMC
June 2021

Spatiotemporal Analysis of B Cell- and Antibody Secreting Cell-Subsets in Human Melanoma Reveals Metastasis-, Tumor Stage-, and Age-Associated Dynamics.

Front Cell Dev Biol 2021 21;9:677944. Epub 2021 May 21.

Laboratory of Molecular Dermato-Oncology and Tumor Immunology, Department of Dermatology, Medical University of Vienna, Vienna, Austria.

The role of tumor-associated B cells in human cancer is only starting to emerge. B cells typically undergo a series of developmental changes in phenotype and function, however, data on the composition of the B cell population in human melanoma are largely absent including changes during tumor progression and their potential clinical significance. In this study, we compared the number and distribution of six major B cell and antibody secreting cell subpopulations outside tertiary lymphoid structures in whole tumor sections of 154 human cutaneous melanoma samples (53 primary tumors without subsequent metastasis, 44 primary tumors with metastasis, 57 metastatic samples) obtained by seven color multiplex immunohistochemistry and automated tissue imaging and analysis. In primary melanomas, we observed the highest numbers for plasmablast-like, memory-like, and activated B cell subtypes. These cells showed a patchy, predominant paratumoral distribution at the invasive tumor-stroma margin. Plasma cell-like cells were hardly detected, germinal center- and transitional/regulatory-like B cells not at all. Of the major clinicopathologic prognostic factors for primary melanomas, metastasis was associated with decreased memory-like B cell numbers and a higher age associated with higher plasmablast-like cell numbers. When we compared the composition of B cell subpopulations in primary melanomas and metastatic samples, we found a significantly higher proportion of plasma cell-like cells at distant metastatic sites and a higher proportion of memory-like B cells at locoregional than distant metastatic sites. Both cell types were detected mainly in the para- and intratumoral stroma. These data provide a first comprehensive and comparative spatiotemporal analysis of major B cell and antibody secreting cell subpopulations in human melanoma and describe metastasis-, tumor stage-, and age-associated dynamics, an important premise for B cell-related biomarker and therapy studies.
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http://dx.doi.org/10.3389/fcell.2021.677944DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8176028PMC
May 2021

B cells sustain inflammation and predict response to immune checkpoint blockade in human melanoma.

Nat Commun 2019 09 13;10(1):4186. Epub 2019 Sep 13.

Department of Dermatology, Medical University of Vienna, 1090, Vienna, Austria.

Tumor associated inflammation predicts response to immune checkpoint blockade in human melanoma. Current theories on regulation of inflammation center on anti-tumor T cell responses. Here we show that tumor associated B cells are vital to melanoma associated inflammation. Human B cells express pro- and anti-inflammatory factors and differentiate into plasmablast-like cells when exposed to autologous melanoma secretomes in vitro. This plasmablast-like phenotype can be reconciled in human melanomas where plasmablast-like cells also express T cell-recruiting chemokines CCL3, CCL4, CCL5. Depletion of B cells in melanoma patients by anti-CD20 immunotherapy decreases tumor associated inflammation and CD8 T cell numbers. Plasmablast-like cells also increase PD-1 T cell activation through anti-PD-1 blockade in vitro and their frequency in pretherapy melanomas predicts response and survival to immune checkpoint blockade. Tumor associated B cells therefore orchestrate and sustain melanoma inflammation and may represent a predictor for survival and response to immune checkpoint blockade therapy.
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http://dx.doi.org/10.1038/s41467-019-12160-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6744450PMC
September 2019

Digital image analysis improves precision of PD-L1 scoring in cutaneous melanoma.

Histopathology 2018 Sep 5;73(3):397-406. Epub 2018 Jun 5.

Cantonal Hospital Baselland, Institute of Pathology, Liestal, Switzerland.

Aims: Immune checkpoint inhibitors have become a successful treatment in metastatic melanoma. The high response rates in a subset of patients suggest that a sensitive companion diagnostic test is required. The predictive value of programmed death ligand 1 (PD-L1) staining in melanoma has been questioned due to inconsistent correlation with clinical outcome. Whether this is due to predictive irrelevance of PD-L1 expression or inaccurate assessment techniques remains unclear. The aim of this study was to develop a standardised digital protocol for the assessment of PD-L1 staining in melanoma and to compare the output data and reproducibility to conventional assessment by expert pathologists.

Methods And Results: In two cohorts with a total of 69 cutaneous melanomas, a highly significant correlation was found between pathologist-based consensus reading and automated PD-L1 analysis (r = 0.97, P < 0.0001). Digital scoring captured the full diagnostic spectrum of PD-L1 expression at single cell resolution. An average of 150 472 melanoma cells (median 38 668 cells; range = 733-1 078 965) were scored per lesion. Machine learning was used to control for heterogeneity introduced by PD-L1-positive inflammatory cells in the tumour microenvironment. The PD-L1 image analysis protocol showed excellent reproducibility (r = 1.0, P < 0.0001) when carried out on independent workstations and reduced variability in PD-L1 scoring of human observers. When melanomas were grouped by PD-L1 expression status, we found a clear correlation of PD-L1 positivity with CD8-positive T cell infiltration, but not with tumour stage, metastasis or driver mutation status.

Conclusion: Digital evaluation of PD-L1 reduces scoring variability and may facilitate patient stratification in clinical practice.
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http://dx.doi.org/10.1111/his.13528DOI Listing
September 2018

Tumor-associated B-cells induce tumor heterogeneity and therapy resistance.

Nat Commun 2017 09 19;8(1):607. Epub 2017 Sep 19.

Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, MA, 02115, USA.

In melanoma, therapies with inhibitors to oncogenic BRAF are highly effective but responses are often short-lived due to the emergence of drug-resistant tumor subpopulations. We describe here a mechanism of acquired drug resistance through the tumor microenvironment, which is mediated by human tumor-associated B cells. Human melanoma cells constitutively produce the growth factor FGF-2, which activates tumor-infiltrating B cells to produce the growth factor IGF-1. B-cell-derived IGF-1 is critical for resistance of melanomas to BRAF and MEK inhibitors due to emergence of heterogeneous subpopulations and activation of FGFR-3. Consistently, resistance of melanomas to BRAF and/or MEK inhibitors is associated with increased CD20 and IGF-1 transcript levels in tumors and IGF-1 expression in tumor-associated B cells. Furthermore, first clinical data from a pilot trial in therapy-resistant metastatic melanoma patients show anti-tumor activity through B-cell depletion by anti-CD20 antibody. Our findings establish a mechanism of acquired therapy resistance through tumor-associated B cells with important clinical implications.Resistance to BRAFV600E inhibitors often occurs in melanoma patients. Here, the authors describe a potential mechanism of acquired drug resistance mediated by tumor-associated B cells-derived IGF-1.
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http://dx.doi.org/10.1038/s41467-017-00452-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5605714PMC
September 2017

Signal Sequence Receptor 2 is required for survival of human melanoma cells as part of an unfolded protein response to endoplasmic reticulum stress.

Mutagenesis 2016 09 14;31(5):573-82. Epub 2016 May 14.

Division of Immunology Allergy and Infectious Diseases (DIAID), Department of Dermatology, Medical University of Vienna, Waehringer Guertel 18-20, Vienna A-1090, Austria,

Current therapy approaches in melanoma targeting have met with the development of resistance and tumour recurrence with a more aggressive phenotype. In a quest for alternative therapy targets, we had previously identified Signal Sequence Receptor 2 (SSR2) as a gene with high expression in a subgroup of human primary melanomas. Now we show that SSR2 exerts a prosurvival functionality in human melanoma cells and that high expression levels of SSR2 are associated with an unfavourable disease outcome in primary melanoma patients. Consistent with SSR's role in translocation of proteins from the ribosome across the endoplasmic reticulum (ER) membrane, our data supports induction of SSR2 as a part of the ER stress response. This response included SSR2 upregulation upon development of therapy resistance to BRAF inhibitors, as well as the dependency of cell survival of BRAF inhibitor-resistant melanoma cells on SSR2. Complementary gain and loss of function data showed the Unfolded Protein Response (UPR) to ER stress as an inducer of SSR2 via transcriptional regulation through X-Box Binding Protein 1s (XBP1s) and support an ER stress-UPR-Transcription Factor XBP1s-SSR2 response axis in human melanocytic cells. Together with its dispensability for survival in normal human cells, these data propose SSR2 as a potential therapeutic target in (therapy-resistant) human melanoma.
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http://dx.doi.org/10.1093/mutage/gew023DOI Listing
September 2016

Tumor-associated B cells in cutaneous primary melanoma and improved clinical outcome.

Hum Pathol 2016 08 21;54:157-64. Epub 2016 Apr 21.

Division of Immunology Allergy and Infectious Diseases (DIAID), Department of Dermatology, Medical University of Vienna, Vienna, Austria, A-1090. Electronic address:

B cells often infiltrate the microenvironment of human tumors. B cells can both positively and negatively regulate antitumor immune responses. In several human cancers, higher numbers of CD20(+) TAB are associated with a favorable prognosis, whereas in human primary melanomas, this association is contentious. In this study, we determined the association of TAB numbers in cutaneous primary melanoma tissue samples and patients' overall survival. The CD20 immunohistochemistry on archival nonmetastasized and metastasized cutaneous primary melanoma tissues from 2 independent patient cohorts was performed. One cohort was used in class comparison for metastasis, the most important prognostic factor for overall survival, and the other cohort for a subsequent survival analysis. Survival association was further validated with RNA data from a third independent cohort. Whole tissue sections were read automatically via quantitative digital imaging and analysis. Survival data were analyzed by Cox proportional hazard modeling. We discovered that cutaneous primary melanomas without metastasis contain significantly more TAB than primary melanomas that had metastasized. At time of first diagnosis, a higher number of TAB is associated with a significantly better overall survival in patients with cutaneous primary melanomas of >1 mm Breslow depth. Also, higher CD20/CD19 tumor mRNA levels are correlated with a significantly better overall survival. Thus, our data support TAB numbers as a prognostic biomarker in cutaneous primary melanoma patients with a tumor of >1 mm Breslow depth. For a survey in larger studies, whole tissue section analysis seems to be key to accurate assessment of TAB numbers.
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http://dx.doi.org/10.1016/j.humpath.2016.03.022DOI Listing
August 2016

The role of tumor microenvironment in melanoma therapy resistance.

Melanoma Manag 2016 Mar 12;3(1):23-32. Epub 2016 Feb 12.

Division of Immunology, Allergy & Infectious Diseases (DIAID), Department of Dermatology, Medical University of Vienna, 1090 Wien, Austria.

Melanoma patients develop resistance to both chemotherapy and targeted-therapy drugs. Promising preclinical and clinical results with immune checkpoint inhibitors using antibodies directed against cytotoxic T-lymphocyte-associated protein 4 and programmed cell death protein 1 have re-energized the field of immune-based therapies in melanoma. However, similar to chemotherapy or targeted therapies, immune checkpoint blockade responds in only subsets of melanoma patients. A number of factors, including gene mutations, altered cell-signaling pathways and tumor heterogeneity can contribute to therapy resistance. Recent studies have highlighted the role of inflammatory tumor microenvironment on therapy resistance of cancer cells. Cancer cells either alone or in conjunction with the tumor stroma can contribute to an inflammatory microenvironment. Multimodal approaches of targeting the tumor microenvironment, in addition to malignant cells, may be necessary for better therapy responses.
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http://dx.doi.org/10.2217/mmt.15.37DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6094607PMC
March 2016

RanBP3 Regulates Melanoma Cell Proliferation via Selective Control of Nuclear Export.

J Invest Dermatol 2016 Jan;136(1):264-74

Division of Immunology Allergy and Infectious Diseases (DIAID), Department of Dermatology, Medical University of Vienna, Vienna, Austria. Electronic address:

Chromosome region maintenance 1-mediated nucleocytoplasmic transport has been shown as a potential anticancer target in various malignancies. However, the role of the most characterized chromosome region maintenance 1 cofactor ran binding protein 3 (RanBP3) in cancer cell biology has never been investigated. Utilizing a loss-of-function experimental setting in a vast collection of genetically varied melanoma cell lines, we observed the requirement of RanBP3 in melanoma cell proliferation and survival. Mechanistically, we suggest the reinstatement of transforming growth factor-β (TGF-β)-Smad2/3-p21(Cip1) tumor-suppressor axis as part of the RanBP3 silencing-associated antiproliferative program. Employing extensive nuclear export sequence analyses and immunofluorescence-based protein localization studies, we further present evidence suggesting the requirement of RanBP3 function for the nuclear exit of the weak nuclear export sequence-harboring extracellular signal-regulated kinase protein, although it is dispensable for general CRM1-mediated nuclear export of strong nuclear export sequence-harboring cargoes. Rendering mechanistic support to RanBP3 silencing-mediated apoptosis, consequent to extracellular signal-regulated kinase nuclear entrapment, we observed increased levels of cytoplasmically restricted nonphosphorylated/active proapoptotic Bcl-2-antagonist of cell death (BAD) protein. Last, we present evidence suggesting the frequently activated mitogen-activated protein kinase signaling in melanoma as a potential founding basis for a deregulated post-translational control of RanBP3 activity. Collectively, the presented data suggest RanBP3 as a potential target for therapeutic intervention in human melanoma.
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http://dx.doi.org/10.1038/JID.2015.401DOI Listing
January 2016

A Peptide to Reduce Pulmonary Edema in a Rat Model of Lung Transplantation.

PLoS One 2015 4;10(11):e0142115. Epub 2015 Nov 4.

Department of Dermatology, Skin and Endothelium Research Division (SERD) Medical University of Vienna, Vienna, Austria.

Background: Despite significant advances in organ preservation, surgical techniques and perioperative care, primary graft dysfunction is a serious medical problem in transplantation medicine in general and a specific problem in patients undergoing lung transplantation. As a result, patients develop lung edema, causing reduced tissue oxygenation capacity, reduced lung compliance and increased requirements for mechanical ventilatory support. Yet, there is no effective strategy available to protect the grafted organ from stress reactions induced by ischemia/reperfusion and by the surgical procedure itself.

Methods: We assessed the effect of a cingulin-derived peptide, XIB13 or a random peptide in an established rat model of allogeneic lung transplantation. Donor lungs and recipients received therapeutic peptide at the time of transplantation and outcome was analyzed 100min and 28 days post grafting.

Results: XIB13 improved blood oxygenation and reduced vascular leak 100min post grafting. Even after 28 days, lung edema was significantly reduced by XIB13 and lungs had reduced fibrotic or necrotic zones. Moreover, the induction of an allogeneic T cell response was delayed indicating a reduced antigen exchange between the donor and the host.

Conclusions: In summary, we provide a new tool to strengthen endothelial barrier function thereby improving outcomes in lung transplantation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0142115PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4633234PMC
June 2016

Comprehensive comparative and semiquantitative proteome of a very low number of native and matched epstein-barr-virus-transformed B lymphocytes infiltrating human melanoma.

J Proteome Res 2014 Jun 6;13(6):2830-45. Epub 2014 May 6.

Division of Immunology, Allergy and Infectious Diseases, Department of Dermatology, Medical University of Vienna , Waehringer Guertel 18-20, A-1090 Vienna, Austria.

Melanoma, the deadliest form of skin cancer, is highly immunogenic and frequently infiltrated with immune cells including B cells. The role of tumor-infiltrating B cells (TIBCs) in melanoma is as yet unresolved, possibly due to technical challenges in obtaining TIBCs in sufficient quantity for extensive studies and due to the limited life span of B cells in vitro. A comprehensive workflow has thus been developed for successful isolation and proteomic analysis of a low number of TIBCs from fresh, human melanoma tissue. In addition, we generated in vitro-proliferating TIBC cultures using simultaneous stimulation with Epstein-Barr virus (EBV) and the TLR9 ligand CpG-oligodesoxynucleotide (CpG ODN). The FASP method and iTRAQ labeling were utilized to obtain a comparative, semiquantitative proteome to assess EBV-induced changes in TIBCs. By using as few as 100 000 B cells (∼5 μg protein)/sample for our proteomic study, a total number of 6507 proteins were identified. EBV-induced changes in TIBCs are similar to those already reported for peripheral B cells and largely involve changes in cell cycle proliferation, apoptosis, and interferon response, while most of the proteins were not significantly altered. This study provides an essential, further step toward detailed characterization of TIBCs including functional in vitro analysis.
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http://dx.doi.org/10.1021/pr401270yDOI Listing
June 2014

MTSS1 is a metastasis driver in a subset of human melanomas.

Nat Commun 2014 Mar 17;5:3465. Epub 2014 Mar 17.

1] Division of Immunology, Allergy and Infectious Diseases (DIAID), Department of Dermatology, Medical University of Vienna, 1090 Vienna, Austria [2] Center for Molecular Medicine (CeMM), Austrian Academy of Sciences, 1090 Vienna, Austria.

In cancers with a highly altered genome, distinct genetic alterations drive subsets rather than the majority of individual tumours. Here we use a sequential search across human tumour samples for transcript outlier data points with associated gene copy number variations that correlate with patient's survival to identify genes with pro-invasive functionality. Employing loss and gain of function approaches in vitro and in vivo, we show that one such gene, MTSS1, promotes the ability of melanocytic cells to metastasize and engages actin dynamics via Rho-GTPases and cofilin in this process. Indeed, high MTSS1 expression defines a subgroup of primary melanomas with unfavourable prognosis. These data underscore the biological, clinical and potential therapeutic implications of molecular subsets within genetically complex cancers.
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http://dx.doi.org/10.1038/ncomms4465DOI Listing
March 2014

NVP-LDE225, a potent and selective SMOOTHENED antagonist reduces melanoma growth in vitro and in vivo.

PLoS One 2013 30;8(7):e69064. Epub 2013 Jul 30.

Division of Immunology, Allergy and Infectious Diseases, Department of Dermatology, Medical University of Vienna, Vienna, Austria.

Melanoma is one of the most aggressive cancers and its incidence is increasing worldwide. So far there are no curable therapies especially after metastasis. Due to frequent mutations in members of the mitogen-activated protein kinase (MAPK) signaling pathway, this pathway is constitutively active in melanoma. It has been shown that the SONIC HEDGEHOG (SHH)-GLI and MAPK signaling pathway regulate cell growth in many tumors including melanoma and interact with each other in the regulation of cell proliferation and survival. Here we show that the SHH-GLI pathway is active in human melanoma cell lines as they express downstream target of this pathway GLI1. Expression of GLI1 was significantly higher in human primary melanoma tissues harboring BRAF(V600E) mutation than those with wild type BRAF. Pharmacologic inhibition of BRAF(V600E) in human melanoma cell lines resulted in decreased expression of GLI1 thus demonstrating interaction of SHH-GLI and MAPK pathways. Inhibition of SHH-GLI pathway by the novel small molecule inhibitor of smoothened NVP-LDE225 was followed by inhibition of cell growth and induction of apoptosis in human melanoma cell lines, interestingly with both BRAF(V600E) and BRAF(Wild Type) status. NVP-LDE225 was potent in reducing cell proliferation and inducing tumor growth arrest in vitro and in vivo, respectively and these effects were superior to the natural compound cyclopamine. Finally, we conclude that inhibition of SHH-GLI signaling pathway in human melanoma by the specific smoothened inhibitor NVP-LDE225 could have potential therapeutic application in human melanoma even in the absence of BRAF(V600E) mutation and warrants further investigations.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0069064PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3728309PMC
April 2014

Suppression of nucleotide metabolism underlies the establishment and maintenance of oncogene-induced senescence.

Cell Rep 2013 Apr 4;3(4):1252-65. Epub 2013 Apr 4.

Gene Expression and Regulation Program, The Wistar Institute Cancer Center, The Wistar Institute, Philadelphia, PA 19104, USA.

Oncogene-induced senescence is characterized by a stable cell growth arrest, thus providing a tumor suppression mechanism. However, the underlying mechanisms for this phenomenon remain unknown. Here, we show that a decrease in deoxyribonucleotide triphosphate (dNTP) levels underlies oncogene-induced stable senescence-associated cell growth arrest. The decrease in dNTP levels is caused by oncogene-induced repression of ribonucleotide reductase subunit M2 (RRM2), a rate-limiting protein in dNTP synthesis. This precedes the senescence-associated cell-cycle exit and coincides with the DNA damage response. Consistently, RRM2 downregulation is both necessary and sufficient for senescence. Strikingly, suppression of nucleotide metabolism by RRM2 repression is also necessary for maintenance of the stable senescence-associated cell growth arrest. Furthermore, RRM2 repression correlates with senescence status in benign nevi and melanoma, and its knockdown drives senescence of melanoma cells. These data reveal the molecular basis whereby the stable growth arrest of oncogene-induced senescence is established and maintained through suppression of nucleotide metabolism.
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http://dx.doi.org/10.1016/j.celrep.2013.03.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3840499PMC
April 2013

Immunotargeting of tumor subpopulations in melanoma patients: A paradigm shift in therapy approaches.

Oncoimmunology 2012 Nov;1(8):1454-1456

Division of Immunology, Allergy and Infectious Diseases; Department of Dermatology; Vienna, Austria.

Several melanoma cell subpopulations with tumor-initiating and/or tumor-maintaining properties exist that may contribute to chemoresistance and tumor recurrence after standard therapies. One of these subpopulations expresses a B-cell marker, CD20. In a small pilot trial, we showed that a subset of Stage IV melanoma patients may potentially benefit from an adjuvant treatment using the anti-CD20 antibody rituximab.
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http://dx.doi.org/10.4161/onci.21357DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3518536PMC
November 2012

Combining filter-aided sample preparation and pseudoshotgun technology to profile the proteome of a low number of early passage human melanoma cells.

J Proteome Res 2013 Feb 24;12(2):1040-8. Epub 2012 Dec 24.

Division of Immunology, Allergy and Infectious Diseases, Department of Dermatology, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria.

The performance of two proteomic sample preparation methods, "pseudoshotgun" (PSG) and filter-aided sample preparation (FASP) were compared in terms of the number of identified proteins, representation of cellular component GO (gene ontology) categories in the obtained list of proteins, and the efficiency of both methods in the proteomic analysis of a very low number of cells. Both methods were combined to obtain a proteomic profile of a short-term culture (passage 3) of melanoma cells, established in our laboratory from a human metastatic melanoma lesion. The data revealed that with FASP, usually more proteins are identified than with PSG when analyzing a higher number of cells (≥ 5000/injection), whereas PSG is favorable when analyzing only a very small amount of cells (250-500/injection). PSG and FASP, however, are complementary techniques, as combining both methods further increases the number of identified proteins. Moreover, we show that it is feasible to identify a substantial number of proteins from only 250 cells/injection that is equivalent to 60 ng of protein.
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http://dx.doi.org/10.1021/pr301009uDOI Listing
February 2013

An attempt at a molecular prediction of metastasis in patients with primary cutaneous melanoma.

PLoS One 2012 14;7(11):e49865. Epub 2012 Nov 14.

Division of Immunology, Allergy and Infectious Diseases, Department of Dermatology, Medical University of Vienna, Vienna, Austria.

Background: Current prognostic clinical and morphological parameters are insufficient to accurately predict metastasis in individual melanoma patients. Several studies have described gene expression signatures to predict survival or metastasis of primary melanoma patients, however the reproducibility among these studies is disappointingly low.

Methodology/principal Findings: We followed extended REMARK/Gould Rothberg criteria to identify gene sets predictive for metastasis in patients with primary cutaneous melanoma. For class comparison, gene expression data from 116 patients with clinical stage I/II (no metastasis) and 72 with III/IV primary melanoma (with metastasis) at time of first diagnosis were used. Significance analysis of microarrays identified the top 50 differentially expressed genes. In an independent data set from a second cohort of 28 primary melanoma patients, these genes were analyzed by multivariate Cox regression analysis and leave-one-out cross validation for association with development of metastatic disease. In a multivariate Cox regression analysis, expression of the genes Ena/vasodilator-stimulated phosphoprotein-like (EVL) and CD24 antigen gave the best predictive value (p = 0.001; p = 0.017, respectively). A multivariate Cox proportional hazards model revealed these genes as a potential independent predictor, which may possibly add (both p = 0.01) to the predictive value of the most important morphological indicator, Breslow depth.

Conclusion/significance: Combination of molecular with morphological information may potentially enable an improved prediction of metastasis in primary melanoma patients. A strength of the gene expression set is the small number of genes, which should allow easy reevaluation in independent data sets and adequately designed clinical trials.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0049865PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3498185PMC
April 2013

Dual suppression of the cyclin-dependent kinase inhibitors CDKN2C and CDKN1A in human melanoma.

J Natl Cancer Inst 2012 Nov 20;104(21):1673-9. Epub 2012 Sep 20.

Division of Immunology, Allergy and Infectious Diseases, Department of Dermatology, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria.

Resistance to BRAF(V600E) inhibitors is associated with reactivation of mitogen-activated protein kinase (MAPK) signaling at different levels in melanoma. To identify downstream effectors of MAPK signaling that could be used as potential additional therapeutic targets for BRAF(V600E) inhibitors, we used hTERT/CDK4R24C/p53DD-immortalized primary human melanocytes genetically modified to ectopically express BRAF ( V600E ) or NRAS ( G12D ) and observed induction of the AP-1 transcription factor family member c-Jun. Using a dominant negative approach, in vitro cell proliferation assays, western blots, and flow cytometry showed that MAPK signaling via BRAF(V600E) promotes melanoma cell proliferation at G1 through AP-1-mediated negative regulation of the INK4 family member, cyclin-dependent kinase inhibitor 2C (CDKN2C), and the CIP/KIP family member, cyclin-dependent kinase inhibitor 1A (CDKN1A). These effects were antagonized by pharmacological inhibition of CDKN2C and CDKN1A targets CDK2 and CDK4 in vitro. In contrast to BRAF ( V600E ) or NRAS ( G12D )-expressing melanocytes, melanoma cells have an inherent resistance to suppression of AP-1 activity by BRAF(V600E)- or MEK-inhibitors. Here, CDK2/4 inhibition statistically significantly augmented the effects of BRAF(V600E)- or MEK-inhibitors on melanoma cell viability in vitro and growth in athymic nude Foxn1 ( nu ) mice (P = .03 when mean tumor volume at day 13 was compared for BRAF(V600E) inhibitor vs BRAF(V600E) inhibitor plus CDK2/4 inhibition; P = .02 when mean tumor volume was compared for MEK inhibitor vs MEK inhibitor plus CDK2/4 inhibition; P values were calculated by a two-sided Welch t test; n = 4-8 mice per group).
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http://dx.doi.org/10.1093/jnci/djs373DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3490842PMC
November 2012

Inhibition of CRM1-mediated nucleocytoplasmic transport: triggering human melanoma cell apoptosis by perturbing multiple cellular pathways.

J Invest Dermatol 2012 Dec 26;132(12):2780-90. Epub 2012 Jul 26.

Division of Immunology, Allergy and Infectious Diseases, Department of Dermatology, Medical University of Vienna, Vienna, Austria.

Development of multiple drug resistance mechanisms in melanomas necessitates the identification of new drug targets, which when inhibited could impact multiple cellular pathways, thus circumventing potential resistance. By performing complementary DNA microarray analysis, we identified four key components of the nucleocytoplasmic transport machinery-CRM1, RAN (RAN-GTPase), RANGAP1, and RANBP1-to be overexpressed in human melanoma metastases. Chromosome region maintenance 1 (CRM1) inhibition induced a marked depletion of prosurvival/cytoplasmic extracellular signal-regulated kinase 1/2 (Erk1/2) and p90 ribosomal S6 kinase1 and elicited persistent Erk-signaling hyperactivation. Consistently, CRM1 inhibition inflicted extensive apoptosis in melanoma cells while sparing nontransformed melanocytes and primary lung fibroblasts. Apoptosis required both the intrinsic and extrinsic apoptotic pathways and was associated with a nuclear entrapment and downregulation of the antiapoptotic CRM1 target protein, Survivin. Apoptosis was preceded by a G1 cell-cycle arrest, and even though CRM1 inhibition mediated marked p53 and p21 induction in wild-type p53 melanoma cells, the latter's silencing or inactivation failed to alleviate apoptosis. Notably, CRM1 inhibition induced cell line-specific, G1 to S progression-retarding changes in the expression of multiple cell-cycle regulatory proteins, thus potentially explaining p53 dispensability. We propose CRM1 as a potential therapeutic target in human melanoma, whose inhibition induces loss of prosurvival/cytoplasmic Erk1/2, mediates persistent Erk hyperactivation, and initiates a multitude of cell context-dependent molecular events to trigger G1 arrest followed by massive apoptosis.
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http://dx.doi.org/10.1038/jid.2012.233DOI Listing
December 2012

A landscape of driver mutations in melanoma.

Cell 2012 Jul;150(2):251-63

The Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA.

Despite recent insights into melanoma genetics, systematic surveys for driver mutations are challenged by an abundance of passenger mutations caused by carcinogenic UV light exposure. We developed a permutation-based framework to address this challenge, employing mutation data from intronic sequences to control for passenger mutational load on a per gene basis. Analysis of large-scale melanoma exome data by this approach discovered six novel melanoma genes (PPP6C, RAC1, SNX31, TACC1, STK19, and ARID2), three of which-RAC1, PPP6C, and STK19-harbored recurrent and potentially targetable mutations. Integration with chromosomal copy number data contextualized the landscape of driver mutations, providing oncogenic insights in BRAF- and NRAS-driven melanoma as well as those without known NRAS/BRAF mutations. The landscape also clarified a mutational basis for RB and p53 pathway deregulation in this malignancy. Finally, the spectrum of driver mutations provided unequivocal genomic evidence for a direct mutagenic role of UV light in melanoma pathogenesis.
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http://dx.doi.org/10.1016/j.cell.2012.06.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3600117PMC
July 2012

Melanoma genome sequencing reveals frequent PREX2 mutations.

Nature 2012 May 9;485(7399):502-6. Epub 2012 May 9.

The Broad Institute of Harvard and MIT, Cambridge, Massachusetts 02142, USA.

Melanoma is notable for its metastatic propensity, lethality in the advanced setting and association with ultraviolet exposure early in life. To obtain a comprehensive genomic view of melanoma in humans, we sequenced the genomes of 25 metastatic melanomas and matched germline DNA. A wide range of point mutation rates was observed: lowest in melanomas whose primaries arose on non-ultraviolet-exposed hairless skin of the extremities (3 and 14 per megabase (Mb) of genome), intermediate in those originating from hair-bearing skin of the trunk (5-55 per Mb), and highest in a patient with a documented history of chronic sun exposure (111 per Mb). Analysis of whole-genome sequence data identified PREX2 (phosphatidylinositol-3,4,5-trisphosphate-dependent Rac exchange factor 2)--a PTEN-interacting protein and negative regulator of PTEN in breast cancer--as a significantly mutated gene with a mutation frequency of approximately 14% in an independent extension cohort of 107 human melanomas. PREX2 mutations are biologically relevant, as ectopic expression of mutant PREX2 accelerated tumour formation of immortalized human melanocytes in vivo. Thus, whole-genome sequencing of human melanoma tumours revealed genomic evidence of ultraviolet pathogenesis and discovered a new recurrently mutated gene in melanoma.
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http://dx.doi.org/10.1038/nature11071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3367798PMC
May 2012

Targeting CD20 in melanoma patients at high risk of disease recurrence.

Mol Ther 2012 May 21;20(5):1056-62. Epub 2012 Feb 21.

Division of Immunology, Allergy and Infectious Diseases, Department of Dermatology, Medical University of Vienna, Vienna, Austria.

Melanomas contain distinct cell subpopulations. Several of these subpopulations, including one expressing CD20, may harbor stem cell-like or tumor-initiating characteristics. We hypothesized that patients at high risk of disease recurrence could benefit from an adjuvant anti-CD20 therapy. Therefore, we initiated a small pilot trial to study the effect of the anti-CD20 antibody rituximab in a group of melanoma patients with stage IV metastatic disease who had been rendered without evident disease by way of surgery, chemotherapy and/or radiation therapy. The major objective was safety, while secondary objectives were description of recurrence-free intervals (RFI) and overall survival (OS). Nine patients received rituximab at 375 mg/m(2) qw for 4 weeks followed by a maintenance therapy every 8 weeks. Treatment was discontinued after 2 years or with disease recurrence. Treatment was well tolerated. After a median observation of 42 months, the median neither of RFI nor of OS has been reached. Despite therapy that ended after 2 years, six out of nine patients are still alive and five of them are recurrence-free. Though the patient number is too small for definitive conclusions, our data may represent a first example of the potential therapeutic value of targeting CD20(+) cell populations-at least for a subset of patients.
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http://dx.doi.org/10.1038/mt.2012.27DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3345981PMC
May 2012

Polo-like kinase 1 is a potential therapeutic target in human melanoma.

J Invest Dermatol 2011 Sep 9;131(9):1886-95. Epub 2011 Jun 9.

Division of Immunology, Allergy and Infectious Diseases, Department of Dermatology, Medical University of Vienna, Vienna, Austria.

Exploration of the human melanoma cell-cycle pathway can lead to identification of new therapeutic targets. By gene set enrichment analysis, we identified the cell-cycle pathway and its member polo-like kinase 1 (Plk-1) to be significantly overexpressed in primary melanomas and in melanoma metastases. In vitro expression of Plk-1 was peaked at the G2/M phase of the cell cycle. Plk-1 knockdown/inhibition led to induction of apoptosis, which was caspase-3/8-dependent and p53-independent, and involved BID and Bcl-2 proteins. Comparative genomic hybridization/single-nucleotide polymorphism arrays showed no genetic alteration in the Plk-1 locus. Previous suggestions and significant enrichment of the mitogen-activated protein kinase (MAPK) signaling pathway pointed to potential regulation of Plk-1 by MAPK signaling. Inhibition of this pathway resulted in decreased Plk-1 expression as a consequence of G1 cell-cycle arrest rather than direct regulation of Plk-1. Inhibition of MAPK and Plk-1 had an additive effect on reduced cell viability. This study shows that in human melanoma, Plk-1 expression is dynamically regulated during the cell cycle, knockdown of Plk-1 leads to apoptotic cell death, and that a combination of Plk-1 and MAPK inhibition has an additive effect on melanoma cell viability. We conclude that combined inhibition of Plk-1 and MAPK could be a potentially attractive strategy in melanoma therapy.
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http://dx.doi.org/10.1038/jid.2011.136DOI Listing
September 2011

Integrative genome comparison of primary and metastatic melanomas.

PLoS One 2010 May 24;5(5):e10770. Epub 2010 May 24.

Department of Medical Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, Massachusetts, USA.

A cardinal feature of malignant melanoma is its metastatic propensity. An incomplete view of the genetic events driving metastatic progression has been a major barrier to rational development of effective therapeutics and prognostic diagnostics for melanoma patients. In this study, we conducted global genomic characterization of primary and metastatic melanomas to examine the genomic landscape associated with metastatic progression. In addition to uncovering three genomic subclasses of metastastic melanomas, we delineated 39 focal and recurrent regions of amplification and deletions, many of which encompassed resident genes that have not been implicated in cancer or metastasis. To identify progression-associated metastasis gene candidates, we applied a statistical approach, Integrative Genome Comparison (IGC), to define 32 genomic regions of interest that were significantly altered in metastatic relative to primary melanomas, encompassing 30 resident genes with statistically significant expression deregulation. Functional assays on a subset of these candidates, including MET, ASPM, AKAP9, IMP3, PRKCA, RPA3, and SCAP2, validated their pro-invasion activities in human melanoma cells. Validity of the IGC approach was further reinforced by tissue microarray analysis of Survivin showing significant increased protein expression in thick versus thin primary cutaneous melanomas, and a progression correlation with lymph node metastases. Together, these functional validation results and correlative analysis of human tissues support the thesis that integrated genomic and pathological analyses of staged melanomas provide a productive entry point for discovery of melanoma metastases genes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0010770PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2875381PMC
May 2010

Induction of targeted cell migration by cutaneous administration of a DNA vector encoding a biologically active chemokine CCL21.

J Invest Dermatol 2010 Jun 25;130(6):1611-23. Epub 2010 Feb 25.

Division of Immunology, Allergy and Infectious Diseases, Department of Dermatology, Medical University of Vienna, Allgemeines Krankenhaus, Währinger Gürtel 18-20, Vienna, Austria.

Skin inflammation can induce local expression of CCL21, which is subsequently drained to lymph nodes (LNs) influencing their cellular composition. To determine whether the same can be achieved by dermal administration of a plasmid DNA (pDNA) encoding CCL21, we generated a pDNA-based gene construct allowing high-level expression of CCL21. Expression and secretion of biologically active CCL21 were confirmed in vitro by immunohistochemistry, western blot analysis, ELISA, and transwell chemotactic assays. In vivo experiments showed cellular expression of transgenic CCL21 after particle-mediated gene gun delivery of pDNA into skin. CCL21 was expressed in the epidermis, consequently secreted into the upper dermis, and transported into the draining LNs, which resulted in increased CCL21 concentration, total cell number, and frequencies of CD11c(+) DCs and CD4(+)/CD62L(+) naïve, CD4(+)/CD62L(-), and CD8(+)/CD62L(-) effector memory T-cells (expressing CCL21 receptors CCR7 or CXCR3), as well as retention of adoptively transferred T-lymphocytes, in the draining LNs of plt/plt mice (lacking endogenous expression of CCL21). Our studies show that biologically active CCL21 can be overexpressed by genetic means in vitro and in vivo. This strategy allows reconstitution of a genetic defect and colocalization of different cell types in the secondary lymphoid organs, an important prerequisite for targeted cell migration.
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http://dx.doi.org/10.1038/jid.2010.31DOI Listing
June 2010

Combination of an EGFR blocker and a COX-2 inhibitor for the treatment of advanced cutaneous squamous cell carcinoma.

J Dtsch Dermatol Ges 2008 Dec;6(12):1066-9

Division of Immunology, Allergy and Infectious Diseases, Department of Dermatology, Medical University of Vienna.

Cutaneous squamous cell carcinoma (SCC) is one of the most common cancers worldwide. Epidermal growth factor receptor (EGFR) is expressed at the cell surface by more than 90% of SCCs and its activation is responsible for cell cycle progression, proliferation, survival, angiogenesis and metastasis. Cyclooxygenase-2 (COX-2) is an enzyme up-regulated through EGFR signaling and responsible for some of the EGFR-dependent biological effects. An 88-year-old man presented with a recurrent, locoregionally meta-static SCC of the right parietal region, which was resistant to radiotherapy. With a combination therapy of an EGFR blocker (cetuximab) and a COX-2 inhibitor (celecoxib), the tumor regressed partially and the patient's Karnofsky index improved. We speculate that the combined use of cetuximab and COX-2 inhibitors can be a new and effective therapy for advanced and recurrent cutaneous SCCs.
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http://dx.doi.org/10.1111/j.1610-0387.2008.06861.xDOI Listing
December 2008

Adjuvant treatment with vindesine in comparison to observation alone in patients with metastasized melanoma after complete metastasectomy: a randomized multicenter trial of the German Dermatologic Cooperative Oncology Group.

Melanoma Res 2008 Oct;18(5):353-8

Department of Dermatology, Skin Cancer Program, Eberhard-Karls-University, Tuebingen, Germany.

To evaluate the efficacy and safety of vindesine in patients with metastatic melanoma after complete metastasectomy. One hundred and forty-two patients with metastatic spread to regional sites, lymph nodes, and distant sites after complete metastasectomy were randomized to receive either treatment with vindesine for 2 years or observation alone. Vindesine 3 mg/m intravenously was administered biweekly for the first 26 weeks following 3-week intervals for an additional 26 weeks and thereafter every 4 weeks for 52 weeks. One hundred and thirty-nine patients were eligible for intent-to-treat analysis. Median follow-up time was 46 months. Median recurrence free survival was 7.9 months in the vindesine group and 7.6 months in the observational group (P=0.40). Three-year overall survival rate was 54.9% (37 patients) for patients receiving vindesine in comparison to 43.6% (31 patients) in the observation arm (P=0.07). No grade IV toxicity was observed. The two major side effects in the vindesine group were alopecia and peripheral neuropathy. Ten patients went off treatment because of grade III toxicity. Adjuvant treatment with vindesine did not significantly prolong disease free or overall survival in high-risk melanoma patients. Thus, this randomized trial did not confirm earlier reports of beneficial effects of adjuvant vindesine and can therefore not be recommended.
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http://dx.doi.org/10.1097/CMR.0b013e32830e3936DOI Listing
October 2008

Gene expression changes in an animal melanoma model correlate with aggressiveness of human melanoma metastases.

Mol Cancer Res 2008 May;6(5):760-9

Howard Hughes Medical Institute, Center for Cancer Research, Cambridge, MA, USA.

Metastasis is the deadliest phase of cancer progression. Experimental models using immunodeficient mice have been used to gain insights into the mechanisms of metastasis. We report here the identification of a "metastasis aggressiveness gene expression signature" derived using human melanoma cells selected based on their metastatic potentials in a xenotransplant metastasis model. Comparison with expression data from human melanoma patients shows that this metastasis gene signature correlates with the aggressiveness of melanoma metastases in human patients. Many genes encoding secreted and membrane proteins are included in the signature, suggesting the importance of tumor-microenvironment interactions during metastasis.
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http://dx.doi.org/10.1158/1541-7786.MCR-07-0344DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2756991PMC
May 2008
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