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1.  Discretization of Gene Expression Data Unmasks Molecular Subgroups Recurring in Different Human Cancer Types 
PLoS ONE  2016;11(8):e0161514.
Despite the individually different molecular alterations in tumors, the malignancy associated biological traits are strikingly similar. Results of a previous study using renal cell carcinoma (RCC) as a model pointed towards cancer-related features, which could be visualized as three groups by microarray based gene expression analysis. In this study, we used a mathematic model to verify the presence of these groups in RCC as well as in other cancer types. We developed an algorithm for gene-expression deviation profiling for analyzing gene expression data of a total of 8397 patients with 13 different cancer types and normal tissues. We revealed three common Cancer Transcriptomic Profiles (CTPs) which recurred in all investigated tumors. Additionally, CTPs remained robust regardless of the functions or numbers of genes analyzed. CTPs may represent common genetic fingerprints, which potentially reflect the closely related biological traits of human cancers.
doi:10.1371/journal.pone.0161514
PMCID: PMC4990327  PMID: 27537329
2.  Characterization of VHL missense mutations in sporadic clear cell renal cell carcinoma: hotspots, affected binding domains, functional impact on pVHL and therapeutic relevance 
BMC Cancer  2016;16:638.
Background
The VHL protein (pVHL) is a multiadaptor protein that interacts with more than 30 different binding partners involved in many oncogenic processes. About 70 % of clear cell renal cell carcinoma (ccRCC) have VHL mutations with varying impact on pVHL function. Loss of pVHL function leads to the accumulation of Hypoxia Inducible Factor (HIF), which is targeted by current targeted treatments. In contrast to nonsense and frameshift mutations that highly likely nullify pVHL multipurpose functions, missense mutations may rather specifically influence the binding capability of pVHL to its partners. The affected pathways may offer predictive clues to therapy and response to treatment. In this study we focused on the VHL missense mutation pattern in ccRCC, and studied their potential effects on pVHL protein stability and binding partners and discussed treatment options.
Methods
We sequenced VHL in 360 sporadic ccRCC FFPE samples and compared observed and expected frequency of missense mutations in 32 different binding domains. The prediction of the impact of those mutations on protein stability and function was assessed in silico. The response to HIF-related, anti-angiogenic treatment of 30 patients with known VHL mutation status was also investigated.
Results
We identified 254 VHL mutations (68.3 % of the cases) including 89 missense mutations (35 %). Codons Ser65, Asn78, Ser80, Trp117 and Leu184 represented hotspots and missense mutations in Trp117 and Leu 184 were predicted to highly destabilize pVHL. About 40 % of VHL missense mutations were predicted to cause severe protein malfunction. The pVHL binding domains for HIF1AN, BCL2L11, HIF1/2α, RPB1, PRKCZ, aPKC-λ/ι, EEF1A1, CCT-ζ-2, and Cullin2 were preferentially affected. These binding partners are mainly acting in transcriptional regulation, apoptosis and ubiquitin ligation. There was no correlation between VHL mutation status and response to treatment.
Conclusions
VHL missense mutations may exert mild, moderate or strong impact on pVHL stability. Besides the HIF binding domain, other pVHL binding sites seem to be non-randomly altered by missense mutations. In contrast to LOF mutations that affect all the different pathways normally controlled by pVHL, missense mutations may be rather appropriate for designing tailor-made treatment strategies for ccRCC.
Electronic supplementary material
The online version of this article (doi:10.1186/s12885-016-2688-0) contains supplementary material, which is available to authorized users.
doi:10.1186/s12885-016-2688-0
PMCID: PMC4987997  PMID: 27530247
Clear cell renal cell carcinoma; VHL; Missense mutations; Binding domains; pVHL stability; Therapy
3.  Clear Cell Papillary Renal Cell Carcinoma and Renal Angiomyoadenomatous Tumor – Two Variants of a Morphologic, Immunohistochemical and Genetic Distinct Entity of Renal Cell Carcinoma 
Clear cell papillary renal cell carcinoma (ccpRCC) and renal angiomyoadenomatous tumor (RAT) share morphologic similarities with clear cell (ccRCC) and papillary renal cell carcinoma (pRCC). It is a matter of controversy whether their morphologic, immunophenotypic and molecular features allow the definition of a separate renal carcinoma entity. The aim of our project was to investigate specific renal immunohistochemical biomarkers involved in the hypoxia-inducible factor pathway and mutations in the VHL gene to clarify the relationship between ccpRCC and RAT. We investigated 28 ccpRCC and 9 RAT samples by immunohistochemistry using 25 markers. VHL gene mutations and allele losses were investigated by Sanger sequencing and fluorescence in situ hybridization (FISH). Clinical follow-up data were obtained for a subset of the patients. No tumor recurrence or tumor-related death was observed in any of the patients. Immunohistochemistry and molecular analyses led to the reclassification of three tumors as ccRCC and TFE3 translocation carcinomas. The immunohistochemical profile of ccpRCC and RAT samples was very similar but not identical, differing from both ccRCC and pRCC. Especially, the parafibromin and hKIM-1 expression exhibited differences in ccpRCC/RAT compared with ccRCC and pRCC. Genetic analysis revealed VHL mutations in 2/27 (7%) and 1/7 (14%) ccpRCC and RAT samples, respectively. FISH analysis disclosed a 3p loss in 2/20 (10 %) ccpRCC samples. ccpRCC and RAT have a specific morphologic and immunohistochemical profile but they share similarities with the more aggressive renal tumors. Based on our results, we regard ccpRCC/RAT as a distinct entity of renal cell carcinomas.
doi:10.1097/PAS.0000000000000456
PMCID: PMC4465996  PMID: 25970683
kidney; clear cell papillary renal cell cancer; cytokeratin 7; VHL; renal angiomyoadenomatous tumor; clear cell renal cell carcinoma
4.  Integrative genome analyses identify key somatic driver mutations of small cell lung cancer 
Peifer, Martin | Fernández-Cuesta, Lynnette | Sos, Martin L | George, Julie | Seidel, Danila | Kasper, Lawryn H | Plenker, Dennis | Leenders, Frauke | Sun, Ruping | Zander, Thomas | Menon, Roopika | Koker, Mirjam | Dahmen, Ilona | Müller, Christian | Di Cerbo, Vincenzo | Schildhaus, Hans-Ulrich | Altmüller, Janine | Baessmann, Ingelore | Becker, Christian | de Wilde, Bram | Vandesompele, Jo | Böhm, Diana | Ansén, Sascha | Gabler, Franziska | Wilkening, Ines | Heynck, Stefanie | Heuckmann, Johannes M | Lu, Xin | Carter, Scott L | Cibulskis, Kristian | Banerji, Shantanu | Getz, Gad | Park, Kwon-Sik | Rauh, Daniel | Grütter, Christian | Fischer, Matthias | Pasqualucci, Laura | Wright, Gavin | Wainer, Zoe | Russell, Prudence | Petersen, Iver | Chen, Yuan | Stoelben, Erich | Ludwig, Corinna | Schnabel, Philipp | Hoffmann, Hans | Muley, Thomas | Brockmann, Michael | Engel-Riedel, Walburga | Muscarella, Lucia A | Fazio, Vito M | Groen, Harry | Timens, Wim | Sietsma, Hannie | Thunnissen, Erik | Smit, Egbert | Heideman, Daniëlle AM | Snijders, Peter JF | Cappuzzo, Federico | Ligorio, Claudia | Damiani, Stefania | Field, John | Solberg, Steinar | Brustugun, Odd Terje | Lund-Iversen, Marius | Sänger, Jörg | Clement, Joachim H | Soltermann, Alex | Moch, Holger | Weder, Walter | Solomon, Benjamin | Soria, Jean-Charles | Validire, Pierre | Besse, Benjamin | Brambilla, Elisabeth | Brambilla, Christian | Lantuejoul, Sylvie | Lorimier, Philippe | Schneider, Peter M | Hallek, Michael | Pao, William | Meyerson, Matthew | Sage, Julien | Shendure, Jay | Schneider, Robert | Büttner, Reinhard | Wolf, Jürgen | Nürnberg, Peter | Perner, Sven | Heukamp, Lukas C | Brindle, Paul K | Haas, Stefan | Thomas, Roman K
Nature genetics  2012;44(10):1104-1110.
Small-cell lung cancer (SCLC) is an aggressive lung tumor subtype with poor survival1–3. We sequenced 29 SCLC exomes, two genomes and 15 transcriptomes and found an extremely high mutation rate of 7.4±1 protein-changing mutations per million basepairs. Therefore, we conducted integrated analyses of the various data sets to identify pathogenetically relevant mutated genes. In all cases we found evidence for inactivation of TP53 and RB1 and identified recurrent mutations in histone-modifying genes, CREBBP, EP300, and MLL. Furthermore, we observed mutations in PTEN, in SLIT2, and EPHA7, as well as focal amplifications of the FGFR1 tyrosine kinase gene. Finally, we detected many of the alterations found in humans in SCLC tumors from p53/Rb1-deficient mice4. Our study implicates histone modification as a major feature of SCLC, reveals potentially therapeutically tractable genome alterations, and provides a generalizable framework for identification of biologically relevant genes in the context of high mutational background.
doi:10.1038/ng.2396
PMCID: PMC4915822  PMID: 22941188
small-cell lung cancer; cancer genome; integrated analysis
5.  Interaction of tumor cells with infiltrating lymphocytes via CD70 and CD27 in clear cell renal cell carcinoma 
Oncoimmunology  2015;4(12):e1049805.
CD70 upregulation by hypoxia-inducible factor and CD27+ lymphocyte tumor infiltration are associated with worse survival in von Hippel-Lindau gene (VHL) mutated clear cell renal cell carcinoma (ccRCC). CD70/CD27 interaction is accompanied by high soluble CD27 levels in the sera of ccRCC patients suggesting that soluble CD27 is a potential predictive tool for anti-CD70 therapy.
doi:10.1080/2162402X.2015.1049805
PMCID: PMC4635726  PMID: 26587319
CD70; CD27; clear cell renal cell carcinoma; soluble CD27; von Hippel-Lindau gene
6.  Interleukin-33 in human gliomas: Expression and prognostic significance 
Oncology Letters  2016;12(1):445-452.
Interleukin-33 (IL-33) is a nuclear and pleiotropic cytokine with regard to its cellular sources and its actions. IL-33 is involved in the pathogenesis of brain diseases. Several factors account for the tumorigenicity of human gliomas, including cytokines and their receptors. The present study assessed the expression and prognostic significance of IL-33 in human astroglial brain tumors. Protein levels of IL-33 were determined by immunohistochemistry using a tissue microarray containing 95 human gliomas. mRNA expression data of IL-33, as well as of its receptors, IL-1 receptor-like 1 protein and IL-1 receptor accessory protein (IL1RAcP), were obtained from The Cancer Genome Atlas database. IL-33 protein was expressed heterogeneously in tumor tissue, but was, however, not detected in normal brain tissue. There was no differential IL-33 protein expression by tumor grade, while IL-33 protein expression was associated with inferior survival in patients with recurrent glioblastomas. Interrogations of the TCGA database indicated that mRNA expression of IL-33 and the IL-33 receptors was heterogeneous, and that IL-33 and IL1RAcP mRNA levels were correlated with the tumor grade. Elevated IL-33 mRNA levels were associated with the inferior survival of glioblastoma patients. Therefore, IL-33 may play an important role in the pathogenesis and prognosis of human gliomas.
doi:10.3892/ol.2016.4626
PMCID: PMC4906635  PMID: 27347163
interleukin-33; interleukin-1 receptor-like 1 protein; interleukin-1 receptor accessory protein; glioma; tissue microarray; survival
7.  Cutaneous Melanoma with Brain Metastasis: Report of 193 Patients with New Observations 
PLoS ONE  2016;11(5):e0156115.
Background
Brain metastasis is a common endpoint in patients suffering from malignant melanoma. However, little is known about factors that predispose to brain metastases.
Objective
We performed a retrospective clinical and pathological investigation of melanoma patients with brain metastases in order to better characterise this patient population.
Methods
193 melanoma patients with brain metastasis histologically diagnosed between 1990 and 2015 at the University Hospital Zurich were retrospectively identified and further specified for sex, age at diagnosis and detection of brain metastasis, and localisation. In addition, data were extracted regarding the subtype of primary melanoma, Breslow tumour thickness, Clark Level, mutation status, extent of metastatic spread and history of a second melanoma.
Results
We found a significant male predominance (n = 126/193; 65%; p < 0.001). Breslow tumour thickness showed a wide range from 0.2 to 12.0 mm (n = 99; median 2.3 mm). 14 of 101 melanomas (14%) were classified as T1, thereof 11 (79%) were found in men. In 32 of 193 patients (17%), the primary melanoma was unknown.
Conclusions
Of special interest in our series is the high incidence of male predominance (79%) in cases of thin metastasing melanoma (14%), implicating genetic or epigenetic (hormonal) gender differences underlying tumour progression. Additionally, the high percentage of unknown primary melanoma (17%), at least partly representing completely regressed melanomas, indicates the importance of immune surveillance in melanoma progression.
doi:10.1371/journal.pone.0156115
PMCID: PMC4877095  PMID: 27213536
8.  Similar lymphocytic infiltration pattern in primary breast cancer and their corresponding distant metastases 
Oncoimmunology  2016;5(6):e1153208.
ABSTRACT
Tumor infiltrating lymphocytes in primary breast cancer (TIL) are acknowledged measures of disease free survival (DFS) in adjuvant and neoadjuvant settings. Little is known about the biology of metastasis infiltrating lymphocytes (mTIL) although the local immunity of the metastatic site may critically influence the infiltrate composite. To address this question, we compared mTIL with their matched TIL in 87 breast cancer patients and their corresponding distant metastasis at four different anatomical locations. Sections of surgical specimen were immunohistochemically analyzed for CD4+, CD8+ and CD20+ lymphocytes in three different tumor compartments: intratumoral lymphocytes (iTIL) defined as lymphocytes in direct contact with breast cancer cells, stromal lymphocytes (sTIL) located within the intratumoral stromal tissue and invasive-margin lymphocytes (imTIL). Overall, we found fewer (p < 0.001) mTIL than TIL. Within the tumor compartments, imTIL were more frequent than sTIL and iTIL both within metastases and the matched primary tumors (PT) (p < 0.001). CD4+ T cells were more numerous than CD8+ T cells and CD20+ B cells (p < 0.001). There was a similar pattern in PT and their corresponding metastasis. Only patients with brain metastases differed from the others displaying less CD20+ B cells at the infiltrative margin of the PT (p < 0.05).
In summary, mTIL were significantly reduced within metastases but still mirrored the infiltrate pattern of the PT, interestingly regardless of the metastatic anatomical locations investigated. Our results suggest that the PT assigns the infiltrating lymphocyte pattern resumed at the metastatic site.
doi:10.1080/2162402X.2016.1153208
PMCID: PMC4938373  PMID: 27471624
Breast cancer; lymphocytes; metastasis; tumor profile
9.  Image-based computational quantification and visualization of genetic alterations and tumour heterogeneity 
Scientific Reports  2016;6:24146.
Recent large-scale genome analyses of human tissue samples have uncovered a high degree of genetic alterations and tumour heterogeneity in most tumour entities, independent of morphological phenotypes and histopathological characteristics. Assessment of genetic copy-number variation (CNV) and tumour heterogeneity by fluorescence in situ hybridization (ISH) provides additional tissue morphology at single-cell resolution, but it is labour intensive with limited throughput and high inter-observer variability. We present an integrative method combining bright-field dual-colour chromogenic and silver ISH assays with an image-based computational workflow (ISHProfiler), for accurate detection of molecular signals, high-throughput evaluation of CNV, expressive visualization of multi-level heterogeneity (cellular, inter- and intra-tumour heterogeneity), and objective quantification of heterogeneous genetic deletions (PTEN) and amplifications (19q12, HER2) in diverse human tumours (prostate, endometrial, ovarian and gastric), using various tissue sizes and different scanners, with unprecedented throughput and reproducibility.
doi:10.1038/srep24146
PMCID: PMC4823793  PMID: 27052161
10.  Cilengitide in newly diagnosed glioblastoma: biomarker expression and outcome 
Oncotarget  2016;7(12):15018-15032.
Integrins αvβ3 and αvβ5 regulate angiogenesis and invasiveness in cancer, potentially by modulating activation of the transforming growth factor (TGF)-β pathway. The randomized phase III CENTRIC and phase II CORE trials explored the integrin inhibitor cilengitide in patients with newly diagnosed glioblastoma with versus without O6-methylguanine DNA methyltransferase (MGMT) promoter methylation. These trials failed to meet their primary endpoints.
Immunohistochemistry was used to assess the levels of the target integrins of cilengitide, αvβ3 and αvβ5 integrins, of αvβ8 and of their putative target, phosphorylation of SMAD2, in tumor tissues from CENTRIC (n=274) and CORE (n=224).
αvβ3 and αvβ5 expression correlated well in tumor and endothelial cells, but showed little association with αvβ8 or pSMAD2 levels. In CENTRIC, there was no interaction between the biomarkers and treatment for prediction of outcome. In CORE, higher αvβ3 levels in tumor cells were associated with improved progression-free survival by central review and with improved overall survival in patients treated with cilengitide.
Integrins αvβ3, αvβ5 and αvβ8 are differentially expressed in glioblastoma. Integrin levels do not correlate with the activation level of the canonical TGF-β pathway. αvβ3 integrin expression may predict benefit from integrin inhibition in patients with glioblastoma lacking MGMT promoter methylation.
doi:10.18632/oncotarget.7588
PMCID: PMC4924768  PMID: 26918452
glioblastoma; integrin; pSmad; TGF-β; biomarker
11.  The Value of In Vitro Diagnostic Testing in Medical Practice: A Status Report 
PLoS ONE  2016;11(3):e0149856.
Background
In vitro diagnostic (IVD) investigations are indispensable for routine patient management. Appropriate testing allows early-stage interventions, reducing late-stage healthcare expenditure (HCE).
Aim
To investigate HCE on IVDs in two developed markets and to assess the perceived value of IVDs on clinical decision-making. Physician-perceived HCE on IVD was evaluated, as well as desired features of new diagnostic markers.
Methods
Past and current HCE on IVD was calculated for the US and Germany. A total of 79 US/German oncologists and cardiologists were interviewed to assess the number of cases where: physicians ask for IVDs; IVDs are used for initial diagnosis, treatment monitoring, or post-treatment; and decision-making is based on an IVD test result. A sample of 201 US and German oncologists and cardiologists was questioned regarding the proportion of HCE they believed to be attributable to IVD testing. After disclosing the actual IVD HCE, the physician’s perception of the appropriateness of the amount was captured. Finally, the association between physician-rated impact of IVD on decision-making and perceived contribution of IVD expenditure on overall HCE was assessed.
Results
IVD costs account for 2.3% and 1.4% of total HCE in the US and Germany. Most physicians (81%) believed that the actual HCE on IVDs was >5%; 19% rated the spending correctly (0–4%, p<0.001). When informed of the actual amount, 64% of physicians rated this as appropriate (p<0.0001); 66% of decision-making was based on IVD. Significantly, more physicians asked for either additional clinical or combined clinical/health economic data than for the product (test/platform) alone (p<0.0001).
Conclusions
Our results indicate a poor awareness of actual HCE on IVD, but a high attributable value of diagnostic procedures for patient management. New markers should deliver actionable and medically relevant information, to guide decision-making and foster improved patient outcomes.
doi:10.1371/journal.pone.0149856
PMCID: PMC4778800  PMID: 26942417
12.  Renal Tumors 
The International Society of Urological Pathology convened a consensus conference on renal cancer, preceded by an online survey, to address issues relating to the diagnosis and reporting of renal neoplasia. In this report, the role of biomarkers in the diagnosis and assessment of prognosis of renal tumors is addressed. In particular we focused upon the use of immunohistochemical markers and the approach to specific differential diagnostic scenarios. We enquired whether cytogenetic and molecular tools were applied in practice and asked for views on the perceived prognostic role of biomarkers. Both the survey and conference voting results demonstrated a high degree of consensus in participants’ responses regarding prognostic/predictive markers and molecular techniques, whereas it was apparent that biomarkers for these purposes remained outside the diagnostic realm pending clinical validation. Although no individual antibody or panel of antibodies reached consensus for classifying renal tumors, or for confirming renal metastatic disease, it was noted from the online survey that 87% of respondents used immunohistochemistry to subtype renal tumors sometimes or occasionally, and a majority (87%) used immunohistochemical markers (Pax 2 or Pax 8, renal cell carcinoma [RCC] marker, panel of pan-CK, CK7, vimentin, and CD10) in confirming the diagnosis of metastatic RCC. There was consensus that immunohistochemistry should be used for histologic subtyping and applied before reaching a diagnosis of unclassified RCC. At the conference, there was consensus that TFE3 and TFEB analysis ought to be requested when RCC was diagnosed in a young patient or when histologic appearances were suggestive of the translocation subtype; whereas Pax 2 and/or Pax 8 were considered to be the most useful markers in the diagnosis of a renal primary.
doi:10.1097/PAS.0b013e318299f12e
PMCID: PMC4714713  PMID: 24025522
renal cell carcinoma; immunohistochemistry; CK7; Pax 2; Pax 8; translocation carcinoma; International Society of Urological Pathology
13.  Oxygen supply maps for hypoxic microenvironment visualization in prostate cancer 
Background:
Intratumoral hypoxia plays an important role with regard to tumor biology and susceptibility to radio- and chemotherapy. For further investigation of hypoxia-related changes, areas of certain hypoxia must be reliably detected within cancer tissues. Pimonidazole, a 2-nitroimindazole, accumulates in hypoxic tissue and can be easily visualized using immunohistochemistry.
Materials and Methods:
To improve detection of highly hypoxic versus normoxic areas in prostate cancer, immunoreactivity of pimonidazole and a combination of known hypoxia-related proteins was used to create computational oxygen supply maps of prostate cancer. Pimonidazole was intravenously administered before radical prostatectomy in n = 15 patients, using the da Vinci robot-assisted surgical system. Prostatectomy specimens were immediately transferred into buffered formaldehyde, fixed overnight, and completely embedded in paraffin. Pimonidazole accumulation and hypoxia-related protein expression were visualized by immunohistochemistry. Oxygen supply maps were created using the normalized information from pimonidazole and hypoxia-related proteins.
Results:
Based on pimonidazole staining and other hypoxia.related proteins (osteopontin, hypoxia-inducible factor 1-alpha, and glucose transporter member 1) oxygen supply maps in prostate cancer were created. Overall, oxygen supply maps consisting of information from all hypoxia-related proteins showed high correlation and mutual information to the golden standard of pimonidazole. Here, we describe an improved computer-based ex vivo model for an accurate detection of oxygen supply in human prostate cancer tissue.
Conclusions:
This platform can be used for precise colocalization of novel candidate hypoxia-related proteins in a representative number of prostate cancer cases, and improve issues of single marker correlations. Furthermore, this study provides a source for further in situ tests and biochemical investigations
doi:10.4103/2153-3539.175376
PMCID: PMC4763504  PMID: 26955501
Hypoxia; pimonidazole; prostate cancer
14.  Connexin 43 expression predicts poor progression-free survival in patients with non-muscle invasive urothelial bladder cancer 
Journal of Clinical Pathology  2015;68(10):819-824.
Objectives
To evaluate the protein expression of connexin 43 (Cx43) in primary urothelial bladder cancer and test its association with the histopathological characteristics and clinical outcome.
Methods
A tissue microarray containing 348 tissue samples from 174 patients with primary urothelial carcinomas of the bladder was immunohistochemically stained for Cx43. The intensity of staining was semiquantitatively evaluated (score 0, 1+, 2+), and the association with clinicopathological features was assessed. Univariable and multivariable analyses were performed to identify predictors for progression-free survival (PFS).
Results
Membranous Cx43 immunoreactivity was detected in 118 (67.8%) of 174 analysable urothelial carcinomas, of which 31 (17.8%) showed even a strong (score 2+) and mainly homogeneous staining. Strong expression levels of Cx43 (score 2+) were associated with higher tumour grade, multiplicity and increased proliferation (all p<0.05). In the subgroup of patients with stage pTa and pT1 bladder tumours (n=158), strong Cx43 expression (p<0.001), solid growth pattern (p<0.001) and increased Ki-67 proliferation fraction (p<0.05) were significantly associated with shorter PFS in an univariable Cox regression analysis. In multivariable Cox regression models, Cx43 immunoreactivity and histological growth pattern remained highly significant and adverse risk factors for PFS.
Conclusions
The expression levels of Cx43 are frequent in non-muscle invasive bladder cancer (NMIBC), with high expression levels being associated with poor prognosis. Routine assessment of Cx43 expression may improve the identification of high-risk NMIBC.
doi:10.1136/jclinpath-2015-202898
PMCID: PMC4602233  PMID: 26251520
TUMOUR MARKERS; URINARY TRACT TUMOURS; BLADDER
15.  Analysis of BRAF and NRAS Mutation Status in Advanced Melanoma Patients Treated with Anti-CTLA-4 Antibodies: Association with Overall Survival? 
PLoS ONE  2015;10(10):e0139438.
Ipilimumab and tremelimumab are human monoclonal antibodies (Abs) against cytotoxic T-lymphocyte antigen-4 (CTLA-4). Ipilimumab was the first agent to show a statistically significant benefit in overall survival in advanced melanoma patients. Currently, there is no proven association between the BRAFV600 mutation and the disease control rate in response to ipilimumab. This analysis was carried out to assess if BRAFV600 and NRAS mutation status affects the clinical outcome of anti-CTLA-4-treated melanoma patients. This is a retrospective multi-center analysis of 101 patients, with confirmed BRAF and NRAS mutation status, treated with anti-CTLA-4 antibodies from December 2006 until August 2012. The median overall survival, defined from the treatment start date with the anti-CTLA-4. Abs-treatment to death or till last follow up, of BRAFV600 or NRAS mutant patients (n = 62) was 10.12 months (95% CI 6.78–13.2) compared to 8.26 months (95% CI 6.02–19.9) in BRAFV600/NRASwt subpopulation (n = 39) (p = 0.67). The median OS of NRAS mutated patients (n = 24) was 12.1 months and although was prolonged compared to the median OS of BRAF mutated patients (n = 38, mOS = 8.03 months) or BRAFV600/NRASwt patients (n = 39, mOS = 8.26 months) the difference didn’t reach statistical significance (p = 0.56). 69 patients were able to complete 4 cycles of anti-CTLA-4 treatment. Of the 24 patients treated with selected BRAF- or MEK-inhibitors, 16 patients received anti-CTLA 4 Abs following either a BRAF or MEK inhibitor with only 8 of them being able to finish 4 cycles of treatment. Based on our results, there is no difference in the median OS in patients treated with anti-CTLA-4 Abs implying that the BRAF/NRAS mutation status alone is not sufficient to predict the outcome of patients treated with anti-CTLA-4 Abs.
doi:10.1371/journal.pone.0139438
PMCID: PMC4591284  PMID: 26426340
16.  The IL-33/ST2 pathway contributes to intestinal tumorigenesis in humans and mice 
Oncoimmunology  2015;5(1):e1062966.
ABSTRACT
Colorectal cancer (CRC) develops through a multistep process and is modulated by inflammation. However, the inflammatory pathways that support intestinal tumors at different stages remain incompletely understood. Interleukin (IL)-33 signaling plays a role in intestinal inflammation, yet its contribution to the pathogenesis of CRC is unknown.
Using immunohistochemistry on 713 resected human CRC specimens, we show here that IL-33 and its receptor ST2 are expressed in low-grade and early-stage human CRCs, and to a lesser extent in higher-grade and more advanced-stage tumors. In a mouse model of CRC, ST2-deficiency protects from tumor development. Moreover, bone marrow (BM) chimera studies indicate that engagement of the IL-33/ST2 pathway on both the radio-resistant and radio-sensitive compartment is essential for CRC development. Mechanistically, activation of IL-33/ST2 signaling compromises the integrity of the intestinal barrier and triggers the production of pro-tumorigenic IL-6 by immune cells.
Together, this data reveals a tumor-promoting role of IL-33/ST2 signaling in CRC.
doi:10.1080/2162402X.2015.1062966
PMCID: PMC4760343  PMID: 26942077
Colorectal cancer; IL-33; inflammation; mouse model; ST2; tissue microarray; tumor microenvironment
17.  Functional characterization of BC039389-GATM and KLK4-KRSP1 chimeric read-through transcripts which are up-regulated in renal cell cancer 
BMC Genomics  2015;16(1):247.
Background
Chimeric read-through RNAs are transcripts originating from two directly adjacent genes (<10 kb) on the same DNA strand. Although they are found in next-generation whole transcriptome sequencing (RNA-Seq) data on a regular basis, investigating them further has usually been refrained from. Therefore, their expression patterns or functions in general, and in oncogenesis in particular, are poorly understood.
Results
We used paired-end RNA-Seq and a specifically designed computational data analysis pipeline (FusionSeq) to nominate read-through events in a small discovery set of renal cell carcinomas (RCC) and confirmed them in a larger validation cohort.
324 read-through events were called overall; 22/27 (81%) selected nominees passed validation with conventional PCR and were sequenced at the junction region. We frequently identified various isoforms of a given read-through event. 2/22 read-throughs were up-regulated: BC039389-GATM was higher expressed in RCC compared to benign adjacent kidney; KLK4-KRSP1 was expressed in 46/169 (27%) RCCs, but rarely in normal tissue. KLK4-KRSP1 expression was associated with worse clinical outcome in the patient cohort. In cell lines, both read-throughs influenced molecular mechanisms (i.e. target gene expression or migration/invasion) in a way that counteracted the effect of the respective parent transcript GATM or KLK4.
Conclusions
Our data suggests that the up-regulation of read-through RNA chimeras in tumors is not random but causes regulatory effects on cellular mechanisms and may impact patient survival.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-015-1446-z) contains supplementary material, which is available to authorized users.
doi:10.1186/s12864-015-1446-z
PMCID: PMC4422297  PMID: 25888189
RNA-Seq; Renal cell carcinoma; Read-through; GATM; KLK4; Chimera
18.  Antagonistic Cross-Regulation between Sox9 and Sox10 Controls an Anti-tumorigenic Program in Melanoma 
PLoS Genetics  2015;11(1):e1004877.
Melanoma is the most fatal skin cancer, but the etiology of this devastating disease is still poorly understood. Recently, the transcription factor Sox10 has been shown to promote both melanoma initiation and progression. Reducing SOX10 expression levels in human melanoma cells and in a genetic melanoma mouse model, efficiently abolishes tumorigenesis by inducing cell cycle exit and apoptosis. Here, we show that this anti-tumorigenic effect functionally involves SOX9, a factor related to SOX10 and upregulated in melanoma cells upon loss of SOX10. Unlike SOX10, SOX9 is not required for normal melanocyte stem cell function, the formation of hyperplastic lesions, and melanoma initiation. To the contrary, SOX9 overexpression results in cell cycle arrest, apoptosis, and a gene expression profile shared by melanoma cells with reduced SOX10 expression. Moreover, SOX9 binds to the SOX10 promoter and induces downregulation of SOX10 expression, revealing a feedback loop reinforcing the SOX10 low/SOX9 high ant,m/ii-tumorigenic program. Finally, SOX9 is required in vitro and in vivo for the anti-tumorigenic effect achieved by reducing SOX10 expression. Thus, SOX10 and SOX9 are functionally antagonistic regulators of melanoma development.
Author Summary
For the development of future cancer therapies it is imperative to understand the molecular processes underlying tumor initiation and expansion. Many key factors involved in these processes have been identified based on cell culture and transplantation experiments, but their relevance for tumor formation and disease progression in the living organism is often unclear. Therefore, genetically modified mice spontaneously developing tumors present indispensable models for cancer research. Here, we address this issue by studying the formation of melanoma, the most fatal skin tumor in industrialized countries. To this end, we use a transgenic mouse model to elucidate cellular and molecular mechanisms regulating congenital nevus and melanoma initiation. We show that a transcription factor called SOX10 promotes melanoma formation by repressing an anti-tumorigenic program involving the activity of a related factor, SOX9. When SOX10 is inactivated, SOX9 becomes upregulated and induces cell cycle arrest and death in melanoma cells. Furthermore, upon experimental elevation of SOX9 levels, SOX10 activity is suppressed, revealing an antagonistic relationship between SOX9 and SOX10 in melanoma initiation. Knowledge of how an anti-tumorigenic program can be stimulated by modulating the activities of these key factors might help to design novel therapeutic strategies.
doi:10.1371/journal.pgen.1004877
PMCID: PMC4309598  PMID: 25629959
19.  Ubiquitylome analysis identifies dysregulation of effector substrates in SPOP-mutant prostate cancer 
Science (New York, N.Y.)  2014;346(6205):85-89.
Cancer genome characterization has revealed driver mutations in genes that govern ubiquitylation; however, the mechanisms by which these alterations promote tumorigenesis remain incompletely characterized. Here, we analyzed changes in the ubiquitin landscape induced by prostate cancer-associated mutations of SPOP, an E3 ubiquitin ligase substrate binding protein. SPOP mutants impaired ubiquitylation of a subset of proteins in a dominant-negative fashion. Of these, DEK and TRIM24 emerged as effector substrates consistently up-regulated by SPOP mutants. We highlight DEK as a SPOP substrate that exhibited decreases in ubiquitylation and proteasomal degradation resulting from heteromeric complexes of wild-type and mutant SPOP protein. DEK stabilization promoted prostate epithelial cell invasion, implicating DEK as an oncogenic effector. More generally, these results provide a framework to decipher tumorigenic mechanisms linked to dysregulated ubiquitylation.
doi:10.1126/science.1250255
PMCID: PMC4257137  PMID: 25278611
20.  Comprehensive MicroRNA Expression Profiling Identifies Novel Markers in Follicular Variant of Papillary Thyroid Carcinoma 
Thyroid  2013;23(11):1383-1389.
Background
Follicular variant of papillary thyroid carcinoma (FVPTC) shares features of papillary (PTC) and follicular (FTC) thyroid carcinomas on a clinical, morphological, and genetic level. MicroRNA (miRNA) deregulation was extensively studied in PTCs and FTCs. However, very limited information is available for FVPTC. The aim of this study was to assess miRNA expression in FVPTC with the most comprehensive miRNA array panel and to correlate it with the clinicopathological data.
Methods
Forty-four papillary thyroid carcinomas (17 FVPTC, 27 classic PTC) and eight normal thyroid tissue samples were analyzed for expression of 748 miRNAs using Human Microarray Assays on the ABI 7900 platform (Life Technologies, Carlsbad, CA). In addition, an independent set of 61 tumor and normal samples was studied for expression of novel miRNA markers detected in this study.
Results
Overall, the miRNA expression profile demonstrated similar trends between FVPTC and classic PTC. Fourteen miRNAs were deregulated in FVPTC with a fold change of more than five (up/down), including miRNAs known to be upregulated in PTC (miR-146b-3p, -146-5p, -221, -222 and miR-222-5p) and novel miRNAs (miR-375, -551b, 181-2-3p, 99b-3p). However, the levels of miRNA expression were different between these tumor types and some miRNAs were uniquely dysregulated in FVPTC allowing separation of these tumors on the unsupervised hierarchical clustering analysis. Upregulation of novel miR-375 was confirmed in a large independent set of follicular cell derived neoplasms and benign nodules and demonstrated specific upregulation for PTC. Two miRNAs (miR-181a-2-3p, miR-99b-3p) were associated with an adverse outcome in FVPTC patients by a Kaplan–Meier (p<0.05) and multivariate Cox regression analysis (p<0.05).
Conclusions
Despite high similarity in miRNA expression between FVPTC and classic PTC, several miRNAs were uniquely expressed in each tumor type, supporting their histopathologic differences. Highly upregulated miRNA identified in this study (miR-375) can serve as a novel marker of papillary thyroid carcinoma, and miR-181a-2-3p and miR-99b-3p can predict relapse-free survival in patients with FVPTC thus potentially providing important diagnostic and predictive value.
doi:10.1089/thy.2012.0632
PMCID: PMC3822383  PMID: 23427895
21.  Concomitant Detection of HER2 Protein and Gene Alterations by Immunohistochemistry (IHC) and Silver Enhanced In Situ Hybridization (SISH) Identifies HER2 Positive Breast Cancer with and without Gene Amplification 
PLoS ONE  2014;9(8):e105961.
Introduction
HER2 status assessment became a mandatory test assay in breast cancer, giving prognostic and predictive information including eligibility for adjuvant anti-HER2 therapy. Precise and reliable assessment of HER2 status is therefore of utmost importance. In this study we analyzed breast cancer samples by a novel technology for concomitant detection of the HER2 protein and gene copy number.
Methods
Tissue microarrays containing 589 invasive breast cancer samples were analyzed with a double immunohistochemistry (IHC) and silver labeled in situ hybridization (SISH) assay simultaneously detecting HER2 protein and gene copy number in the same tumor cells. This bright-field assay was analyzed using scores according to the modified ASCO guidelines and the results were correlated with patient prognosis.
Results
Overall concordance rate between protein expression and the presence of gene amplification was 98%. Fifty-seven of 60 tumors (95%) with IHC score 3+, 6 of 10 tumors with IHC score 2+ (60%) and only 3 of 519 tumors (0.6%) with IHC score 0/1+ were amplified by SISH. Patients with gene amplification despite IHC score 0/1+ had a tendency for worse overall survival (p = 0.088, reaching nearly statistical significance) compared to IHC score 0/1+ without amplification. In contrast, there was no difference in overall survival in IHC score 3+/2+ tumors with and without gene amplification.
Conclusions
The novel double IHC and SISH assay for HER2 is efficient in the identification of breast cancer with discordant HER2 protein and HER2 gene status, especially for the prognostically relevant groups of HER2 protein negative tumors with HER2 amplification and HER2 protein positive tumors without HER2 amplification. Breast cancer without HER2 amplification among IHC score 2+/3+ tumors (10% in our cohort) suggests that other mechanisms than gene amplification contribute to protein overexpression in these cells.
doi:10.1371/journal.pone.0105961
PMCID: PMC4143343  PMID: 25153153
22.  MicroRNA profile of poorly differentiated thyroid carcinomas – new diagnostic and prognostic insights 
The diagnosis of conventional and oncocytic poorly differentiated thyroid carcinomas is difficult. The aim of this study was to characterize their largely unknown miRNA expression profile and to compare it to well differentiated thyroid tumors as well as to identify miRNAs which could potentially serve as diagnostic and prognostic markers.
A total of 14 poorly differentiated, 13 oncocytic poorly differentiated, 72 well differentiated thyroid carcinomas and 8 normal thyroid specimens were studied for expression of 768 miRNAs using PCR-Microarrays.
MiRNA expression was different between poorly differentiated and oncocytic poorly differentiated thyroid carcinomas demonstrating individual clusters on the clustering analysis. Both tumor types showed upregulation of miR-125a-5p, -15a-3p, -182, -183-3p, -222, -222-5p and downregulation of miR-130b, -139-5p, -150, -193a-5p, -219-5p, -23b, -451, -455-3p and of miR-886-3p as compared to normal thyroid tissue. In addition, the oncocytic poorly differentiated thyroid carcinomas demonstrated upregulation of miR-221 and miR-885-5p. The difference in expression was also observed between miRNA expression in poorly differentiated and well differentiated tumors. The CHAID algorithm allowed to separate poorly differentiated from well differentiated thyroid carcinomas with a 73–79% accuracy using miR-23b and miR-150 as a separator. Kaplan-Meier and multivariate analysis showed a significant association with tumor relapses (for miR-23b) and with tumor specific death (for miR-150) in poorly differentiated and oncocytic poorly differentiated thyroid carcinomas.
MiRNA expression is different in conventional and oncocytic poorly differentiated thyroid carcinomas in comparison to well differentiated thyroid cancers and can be used for discrimination between these tumor types. The newly identified deregulated miRNAs (miR-150, miR-23b) bear the potential to be used in a clinical setting delivering prognostic and diagnostic information.
doi:10.1530/JME-13-0266
PMCID: PMC4010646  PMID: 24443580
thyroid cancer; poorly differentiated carcinoma carcinoma; follicular thyroid carcinoma; papillary carcinoma; miRNA profiling; diagnosis; prognosis
23.  Modelling of a genetically diverse evolution of Systemic Mastocytosis with Chronic Myelomonocytic Leukemia (SM-CMML) by Next Generation Sequencing 
Background
Systemic mastocytosis (SM) is a heterogenous, clonal mast cell (MC) proliferation, rarely associated with clonal hematologic non-mast cell lineage disease (SM-AHNMD). KITD816V is regarded as driver-mutation in SM-AHNMD.
Methods
DNA isolated from peripheral blood (PB) of an SM-CMML patient was investigated with targeted next generation sequencing. Variants were verified by Sanger sequencing and further characterized in the SM part of the bone marrow trephine (BMT), normal tissue, and FACS sorted PB cell subpopulations.
Findings
Low coverage deep-sequencing (mean 10x) on a GS 454 Junior revealed two as yet unreported SNVs (CBFA2T3 and CLTCL1), both germ-line mutations. High coverage (mean 1674x) targeted re-sequencing on an Ion Proton revealed 177 variants in coding regions. Excluding SNPs, the final list comprised 11 variants. Among these, TET2 (p.Thr1027fs, p.Cys1263Ser) and RUNX1 (p.Asn109Ser) were identified in in the peripheral blood and the SM part of BMT, but not in normal tissue. Furthermore, Sanger sequencing of PB cells revealed similar signal intensities for both TET2 mutations in FACS sorted CD34+ precursor cells and CD16+ granulocytes comparable to signals in the SM part of BMT. In contrast, RUNX1 exhibited a double intensity in CD34+ cells compared to the SM part of BMT and a homozygous variant signal in granulocytes. Both TET2 and RUNX1 mutations were not detectable in B- and T-cells.
Conclusion
We present a heterozygous triple-mutation pattern (KIT, TET2, RUNX1) in mast cells (SM disease part) with additional LOH of RUNX1 in granulocytes (CMML disease part). These identified mutations allow a more detailed insight into a multistep pathogenesis which suggests a common tumor progenitor in SM-CMML.
doi:10.1186/2162-3619-3-18
PMCID: PMC4100747  PMID: 25032071
Chronic myelomonocytic leukemia; Systemic mastocytosis; SM-CMML; Next Generation Sequencing; c-KIT mutation; TET-2 mutation; RUNX1 mutation
24.  Effect of MRE11 Loss on PARP-Inhibitor Sensitivity in Endometrial Cancer In Vitro 
PLoS ONE  2014;9(6):e100041.
Aim of the study
To evaluate the frequency of MRE11/RAD50/NBS1 (MRN)-complex loss of protein expression in endometrial cancers (EC) and to determine whether loss of MRE11 renders the cancer cells sensitive to Poly(ADP-ribose) polymerase (PARP)-inhibitory treatment.
Methods
MRN expression was examined in 521 samples of endometrial carcinomas and in 10 cancer cell lines. A putative mutation hotspot in the form of an intronic poly(T) allele in MRE11 was sequenced in selected cases (n = 26). Sensitivity to the PARP-inhibitor, BMN673 was tested in colony formation assays before and after MRE11 silencing using siRNA. Homologous recombination (HR) DNA repair was evaluated by RAD51-foci formation assay upon irradiation and drug treatment.
Results
Loss of MRE11 protein was found in 30.7% of EC tumours and significantly associated with loss of RAD50, NBS1 and mismatch repair protein expression. One endometrial cell line showed a markedly reduced MRE11 expression due to a homozygous poly(T) mutation of MRE11, thereby exhibiting an increased sensitivity to BMN673. MRE11 depletion sensitizes MRE11 expressing EC cell lines to the treatment with BMN673. The increased sensitivity to PARP-inhibition correlates with reduced RAD51 foci formation upon ionizing radiation in MRE11-depleted cells.
Conclusion
Loss of the MRE11 protein predicts sensitivity to PARP-inhibitor sensitivity in vitro, defining it as an additional synthetic lethal gene with PARP. The high incidence of MRE11 loss in ECs can be potentially exploited for PARP-inhibitor therapy. Furthermore, MRE11 protein expression using immunohistochemistry could be investigated as a predictive biomarker for PARP-inhibitor treatment.
doi:10.1371/journal.pone.0100041
PMCID: PMC4057395  PMID: 24927325
25.  Automated Assessment of β-Cell Area and Density per Islet and Patient Using TMEM27 and BACE2 Immunofluorescence Staining in Human Pancreatic β-Cells 
PLoS ONE  2014;9(6):e98932.
In this study we aimed to establish an unbiased automatic quantification pipeline to assess islet specific features such as β-cell area and density per islet based on immunofluorescence stainings. To determine these parameters, the in vivo protein expression levels of TMEM27 and BACE2 in pancreatic islets of 32 patients with type 2 diabetes (T2D) and in 28 non-diabetic individuals (ND) were used as input for the automated pipeline. The output of the automated pipeline was first compared to a previously developed manual area scoring system which takes into account the intensity of the staining as well as the percentage of cells which are stained within an islet. The median TMEM27 and BACE2 area scores of all islets investigated per patient correlated significantly with the manual scoring and with the median area score of insulin. Furthermore, the median area scores of TMEM27, BACE2 and insulin calculated from all T2D were significantly lower compared to the one of all ND. TMEM27, BACE2, and insulin area scores correlated as well in each individual tissue specimen. Moreover, islet size determined by costaining of glucagon and either TMEM27 or BACE2 and β-cell density based either on TMEM27 or BACE2 positive cells correlated significantly. Finally, the TMEM27 area score showed a positive correlation with BMI in ND and an inverse pattern in T2D. In summary, automated quantification outperforms manual scoring by reducing time and individual bias. The simultaneous changes of TMEM27, BACE2, and insulin in the majority of the β–cells suggest that these proteins reflect the total number of functional insulin producing β–cells. Additionally, β–cell subpopulations may be identified which are positive for TMEM27, BACE2 or insulin only. Thus, the cumulative assessment of all three markers may provide further information about the real β–cell number per islet.
doi:10.1371/journal.pone.0098932
PMCID: PMC4048234  PMID: 24905913

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