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author:("höfle, Heinz")
1.  Analysis of mammalian gene function through broad based phenotypic screens across a consortium of mouse clinics 
de Angelis, Martin Hrabě | Nicholson, George | Selloum, Mohammed | White, Jacqui | Morgan, Hugh | Ramirez-Solis, Ramiro | Sorg, Tania | Wells, Sara | Fuchs, Helmut | Fray, Martin | Adams, David J | Adams, Niels C | Adler, Thure | Aguilar-Pimentel, Antonio | Ali-Hadji, Dalila | Amann, Gregory | André, Philippe | Atkins, Sarah | Auburtin, Aurelie | Ayadi, Abdel | Becker, Julien | Becker, Lore | Bedu, Elodie | Bekeredjian, Raffi | Birling, Marie-Christine | Blake, Andrew | Bottomley, Joanna | Bowl, Mike | Brault, Véronique | Busch, Dirk H | Bussell, James N | Calzada-Wack, Julia | Cater, Heather | Champy, Marie-France | Charles, Philippe | Chevalier, Claire | Chiani, Francesco | Codner, Gemma F | Combe, Roy | Cox, Roger | Dalloneau, Emilie | Dierich, André | Di Fenza, Armida | Doe, Brendan | Duchon, Arnaud | Eickelberg, Oliver | Esapa, Chris T | El Fertak, Lahcen | Feigel, Tanja | Emelyanova, Irina | Estabel, Jeanne | Favor, Jack | Flenniken, Ann | Gambadoro, Alessia | Garrett, Lilian | Gates, Hilary | Gerdin, Anna-Karin | Gkoutos, George | Greenaway, Simon | Glasl, Lisa | Goetz, Patrice | Da Cruz, Isabelle Goncalves | Götz, Alexander | Graw, Jochen | Guimond, Alain | Hans, Wolfgang | Hicks, Geoff | Hölter, Sabine M | Höfler, Heinz | Hancock, John M | Hoehndorf, Robert | Hough, Tertius | Houghton, Richard | Hurt, Anja | Ivandic, Boris | Jacobs, Hughes | Jacquot, Sylvie | Jones, Nora | Karp, Natasha A | Katus, Hugo A | Kitchen, Sharon | Klein-Rodewald, Tanja | Klingenspor, Martin | Klopstock, Thomas | Lalanne, Valerie | Leblanc, Sophie | Lengger, Christoph | le Marchand, Elise | Ludwig, Tonia | Lux, Aline | McKerlie, Colin | Maier, Holger | Mandel, Jean-Louis | Marschall, Susan | Mark, Manuel | Melvin, David G | Meziane, Hamid | Micklich, Kateryna | Mittelhauser, Christophe | Monassier, Laurent | Moulaert, David | Muller, Stéphanie | Naton, Beatrix | Neff, Frauke | Nolan, Patrick M | Nutter, Lauryl MJ | Ollert, Markus | Pavlovic, Guillaume | Pellegata, Natalia S | Peter, Emilie | Petit-Demoulière, Benoit | Pickard, Amanda | Podrini, Christine | Potter, Paul | Pouilly, Laurent | Puk, Oliver | Richardson, David | Rousseau, Stephane | Quintanilla-Fend, Leticia | Quwailid, Mohamed M | Racz, Ildiko | Rathkolb, Birgit | Riet, Fabrice | Rossant, Janet | Roux, Michel | Rozman, Jan | Ryder, Ed | Salisbury, Jennifer | Santos, Luis | Schäble, Karl-Heinz | Schiller, Evelyn | Schrewe, Anja | Schulz, Holger | Steinkamp, Ralf | Simon, Michelle | Stewart, Michelle | Stöger, Claudia | Stöger, Tobias | Sun, Minxuan | Sunter, David | Teboul, Lydia | Tilly, Isabelle | Tocchini-Valentini, Glauco P | Tost, Monica | Treise, Irina | Vasseur, Laurent | Velot, Emilie | Vogt-Weisenhorn, Daniela | Wagner, Christelle | Walling, Alison | Weber, Bruno | Wendling, Olivia | Westerberg, Henrik | Willershäuser, Monja | Wolf, Eckhard | Wolter, Anne | Wood, Joe | Wurst, Wolfgang | Yildirim, Ali Önder | Zeh, Ramona | Zimmer, Andreas | Zimprich, Annemarie | Holmes, Chris | Steel, Karen P | Herault, Yann | Gailus-Durner, Valérie | Mallon, Ann-Marie | Brown, Steve DM
Nature genetics  2015;47(9):969-978.
The function of the majority of genes in the mouse and human genomes remains unknown. The mouse ES cell knockout resource provides a basis for characterisation of relationships between gene and phenotype. The EUMODIC consortium developed and validated robust methodologies for broad-based phenotyping of knockouts through a pipeline comprising 20 disease-orientated platforms. We developed novel statistical methods for pipeline design and data analysis aimed at detecting reproducible phenotypes with high power. We acquired phenotype data from 449 mutant alleles, representing 320 unique genes, of which half had no prior functional annotation. We captured data from over 27,000 mice finding that 83% of the mutant lines are phenodeviant, with 65% demonstrating pleiotropy. Surprisingly, we found significant differences in phenotype annotation according to zygosity. Novel phenotypes were uncovered for many genes with unknown function providing a powerful basis for hypothesis generation and further investigation in diverse systems.
PMCID: PMC4564951  PMID: 26214591
2.  Variation in Cell Signaling Protein Expression May Introduce Sampling Bias in Primary Epithelial Ovarian Cancer 
PLoS ONE  2013;8(10):e77825.
Although the expression of cell signaling proteins is used as prognostic and predictive biomarker, variability of protein levels within tumors is not well studied. We assessed intratumoral heterogeneity of protein expression within primary ovarian cancer. Full-length proteins were extracted from 88 formalin-fixed and paraffin-embedded tissue samples of 13 primary high-grade serous ovarian carcinomas with 5–9 samples each. In addition, 14 samples of normal fallopian tube epithelium served as reference. Quantitative reverse phase protein arrays were used to analyze the expression of 36 cell signaling proteins including HER2, EGFR, PI3K/Akt, and angiogenic pathways as well as 15 activated (phosphorylated) proteins. We found considerable intratumoral heterogeneity in the expression of proteins with a mean coefficient of variation of 25% (range 17–53%). The extent of intratumoral heterogeneity differed between proteins (p<0.005). Interestingly, there were no significant differences in the extent of heterogeneity between phosphorylated and non-phosphorylated proteins. In comparison, we assessed the variation of protein levels amongst tumors from different patients, which revealed a similar mean coefficient of variation of 21% (range 12–48%). Based on hierarchical clustering, samples from the same patient clustered more closely together compared to samples from different patients. However, a clear separation of tumor versus normal tissue by clustering was only achieved when mean expression values of all individual samples per tumor were analyzed. While differential expression of some proteins was detected independently of the sampling method used, the majority of proteins only demonstrated differential expression when mean expression values of multiple samples per tumor were analyzed. Our data indicate that assessment of established and novel cell signaling proteins as diagnostic or prognostic markers may require sampling of serous ovarian cancers at several distinct locations to avoid sampling bias.
PMCID: PMC3810127  PMID: 24204986
3.  Rapamycin extends murine lifespan but has limited effects on aging 
The Journal of Clinical Investigation  2013;123(8):3272-3291.
Aging is a major risk factor for a large number of disorders and functional impairments. Therapeutic targeting of the aging process may therefore represent an innovative strategy in the quest for novel and broadly effective treatments against age-related diseases. The recent report of lifespan extension in mice treated with the FDA-approved mTOR inhibitor rapamycin represented the first demonstration of pharmacological extension of maximal lifespan in mammals. Longevity effects of rapamycin may, however, be due to rapamycin’s effects on specific life-limiting pathologies, such as cancers, and it remains unclear if this compound actually slows the rate of aging in mammals. Here, we present results from a comprehensive, large-scale assessment of a wide range of structural and functional aging phenotypes, which we performed to determine whether rapamycin slows the rate of aging in male C57BL/6J mice. While rapamycin did extend lifespan, it ameliorated few studied aging phenotypes. A subset of aging traits appeared to be rescued by rapamycin. Rapamycin, however, had similar effects on many of these traits in young animals, indicating that these effects were not due to a modulation of aging, but rather related to aging-independent drug effects. Therefore, our data largely dissociate rapamycin’s longevity effects from effects on aging itself.
PMCID: PMC3726163  PMID: 23863708
4.  The impact of Cysteine-Rich Intestinal Protein 1 (CRIP1) in human breast cancer 
Molecular Cancer  2013;12:28.
CRIP1 (cysteine-rich intestinal protein 1) has been found in several tumor types, its prognostic impact and its role in cellular processes, particularly in breast cancer, are still unclear.
To elucidate the prognostic impact of CRIP1, we analyzed tissues from 113 primary invasive ductal breast carcinomas using immunohistochemistry. For the functional characterization of CRIP1, its endogenous expression was transiently downregulated in T47D and BT474 breast cancer cells and the effects analyzed by immunoblotting, WST-1 proliferation assay and invasion assay.
We found a significant correlation between CRIP1 and HER2 (human epidermal growth factor receptor 2) expression levels (p = 0.016) in tumor tissues. In Kaplan Meier analyses, CRIP1 expression was significantly associated with the distant metastases-free survival of patients, revealing a better prognosis for high CRIP1 expression (p = 0.039). Moreover, in multivariate survival analyses, the expression of CRIP1 was an independent negative prognostic factor, along with the positive prognosticators nodal status and tumor size (p = 0.029). CRIP1 knockdown in the T47D and BT474 breast cancer cell lines led to the increased phosphorylation of MAPK and Akt, to the reduced phosphorylation of cdc2, and to a significantly elevated cell proliferation in vitro (p < 0.001). These results indicate that reduced CRIP1 levels may increase cell proliferation and activate cell growth. In addition, CRIP1 knockdown increased cell invasion in vitro.
Because the lack of CRIP1 expression in breast cancer tissue is significantly associated with a worse prognosis for patients and low endogenous CRIP1 levels in vitro increased the malignant potential of breast cancer cells, we hypothesize that CRIP1 may act as a tumor suppressor in proliferation and invasion processes. Therefore, CRIP1 may be an independent prognostic marker with significant predictive power for use in breast cancer therapy.
PMCID: PMC3666946  PMID: 23570421
CRIP1; HER2; ERB-B2; Breast cancer; Tumorigenesis; Prognosis; Invasion; RNAi; RNA interference; Proliferation
5.  Expression Profiling of Stem Cell-Related Genes in Neoadjuvant-Treated Gastric Cancer: A NOTCH2, GSK3B and β-catenin Gene Signature Predicts Survival 
PLoS ONE  2012;7(9):e44566.
Cancer stem cell (CSC) based gene expression signatures are associated with prognosis in various tumour types and CSCs are suggested to be particularly drug resistant. The aim of our study was first, to determine the prognostic significance of CSC-related gene expression in residual tumour cells of neoadjuvant-treated gastric cancer (GC) patients. Second, we wished to examine, whether expression alterations between pre- and post-therapeutic tumour samples exist, consistent with an enrichment of drug resistant tumour cells. The expression of 44 genes was analysed in 63 formalin-fixed, paraffin embedded tumour specimens with partial tumour regression (10–50% residual tumour) after neoadjuvant chemotherapy by quantitative real time PCR low-density arrays. A signature of combined GSK3Bhigh, β-catenin (CTNNB1)high and NOTCH2low expression was strongly correlated with better patient survival (p<0.001). A prognostic relevance of these genes was also found analysing publically available gene expression data. The expression of 9 genes was compared between pre-therapeutic biopsies and post-therapeutic resected specimens. A significant post-therapeutic increase in NOTCH2, LGR5 and POU5F1 expression was found in tumours with different tumour regression grades. No significant alterations were observed for GSK3B and CTNNB1. Immunohistochemical analysis demonstrated a chemotherapy-associated increase in the intensity of NOTCH2 staining, but not in the percentage of NOTCH2. Taken together, the GSK3B, CTNNB1 and NOTCH2 expression signature is a novel, promising prognostic parameter for GC. The results of the differential expression analysis indicate a prominent role for NOTCH2 and chemotherapy resistance in GC, which seems to be related to an effect of the drugs on NOTCH2 expression rather than to an enrichment of NOTCH2 expressing tumour cells.
PMCID: PMC3438181  PMID: 22970250
6.  Innovations in phenotyping of mouse models in the German Mouse Clinic 
Mammalian Genome  2012;23(9-10):611-622.
Under the label of the German Mouse Clinic (GMC), a concept has been developed and implemented that allows the better understanding of human diseases on the pathophysiological and molecular level. This includes better understanding of the crosstalk between different organs, pleiotropy of genes, and the systemic impact of envirotypes and drugs. In the GMC, experts from various fields of mouse genetics and physiology, in close collaboration with clinicians, work side by side under one roof. The GMC is an open-access platform for the scientific community by providing phenotypic analysis in bilateral collaborations (“bottom-up projects”) and as a partner and driver in international large-scale biology projects (“top-down projects”). Furthermore, technology development is a major topic in the GMC. Innovative techniques for primary and secondary screens are developed and implemented into the phenotyping pipelines (e.g., detection of volatile organic compounds, VOCs).
PMCID: PMC3463795  PMID: 22926221
7.  Evidence of Prognostic Relevant Expression Profiles of Heat-Shock Proteins and Glucose-Regulated Proteins in Oesophageal Adenocarcinomas 
PLoS ONE  2012;7(7):e41420.
A high percentage of oesophageal adenocarcinomas show an aggressive clinical behaviour with a significant resistance to chemotherapy. Heat-shock proteins (HSPs) and glucose-regulated proteins (GRPs) are molecular chaperones that play an important role in tumour biology. Recently, novel therapeutic approaches targeting HSP90/GRP94 have been introduced for treating cancer. We performed a comprehensive investigation of HSP and GRP expression including HSP27, phosphorylated (p)-HSP27(Ser15), p-HSP27(Ser78), p-HSP27(Ser82), HSP60, HSP70, HSP90, GRP78 and GRP94 in 92 primary resected oesophageal adenocarcinomas by using reverse phase protein arrays (RPPA), immunohistochemistry (IHC) and real-time quantitative RT-PCR (qPCR). Results were correlated with pathologic features and survival. HSP/GRP protein and mRNA expression was detected in all tumours at various levels. Unsupervised hierarchical clustering showed two distinct groups of tumours with specific protein expression patterns: The hallmark of the first group was a high expression of p-HSP27(Ser15, Ser78, Ser82) and low expression of GRP78, GRP94 and HSP60. The second group showed the inverse pattern with low p-HSP27 and high GRP78, GRP94 and HSP60 expression. The clinical outcome for patients from the first group was significantly improved compared to patients from the second group, both in univariate analysis (p = 0.015) and multivariate analysis (p = 0.029). Interestingly, these two groups could not be distinguished by immunohistochemistry or qPCR analysis. In summary, two distinct and prognostic relevant HSP/GRP protein expression patterns in adenocarcinomas of the oesophagus were detected by RPPA. Our approach may be helpful for identifying candidates for specific HSP/GRP-targeted therapies.
PMCID: PMC3404067  PMID: 22911792
8.  Common Protein Biomarkers Assessed by Reverse Phase Protein Arrays Show Considerable Intratumoral Heterogeneity in Breast Cancer Tissues 
PLoS ONE  2012;7(7):e40285.
Proteins are used as prognostic and predictive biomarkers in breast cancer. However, the variability of protein expression within the same tumor is not well studied. The aim of this study was to assess intratumoral heterogeneity in protein expression levels by reverse-phase-protein-arrays (RPPA) (i) within primary breast cancers and (ii) between axillary lymph node metastases from the same patient. Protein was extracted from 106 paraffin-embedded samples from 15 large (≥3 cm) primary invasive breast cancers, including different zones within the primary tumor (peripheral, intermediate, central) as well as 2–5 axillary lymph node metastases in 8 cases. Expression of 35 proteins including 15 phosphorylated proteins representing the HER2, EGFR, and uPA/PAI-1 signaling pathways was assessed using reverse-phase-protein-arrays. All 35 proteins showed considerable intratumoral heterogeneity within primary breast cancers with a mean coefficient of variation (CV) of 31% (range 22–43%). There were no significant differences between phosphorylated (CV 32%) and non-phosphorylated proteins (CV 31%) and in the extent of intratumoral heterogeneity within a defined tumor zone (CV 28%, range18–38%) or between different tumor zones (CV 24%, range 17–38%). Lymph node metastases from the same patient showed a similar heterogeneity in protein expression (CV 27%, range 18–34%). In comparison, the variation amongst different patients was higher in primary tumors (CV 51%, range 29–98%) and lymph node metastases (CV 65%, range 40–146%). Several proteins showed significant differential expression between different tumor stages, grades, histological subtypes and hormone receptor status. Commonly used protein biomarkers of breast cancer, including proteins from HER2, uPA/PAI-1 and EGFR signaling pathways showed higher than previously reported intratumoral heterogeneity of expression levels both within primary breast cancers and between lymph node metastases from the same patient. Assessment of proteins as diagnostic or prognostic markers may require tumor sampling in several distinct locations to avoid sampling bias.
PMCID: PMC3390380  PMID: 22792263
9.  uPA and PAI-1-Related Signaling Pathways Differ between Primary Breast Cancers and Lymph Node Metastases12 
Translational Oncology  2012;5(2):98-104.
The supporting role of urokinase-type plasminogen activator (uPA) and its inhibitor plasminogen activator inhibitor 1 (PAI-1) in migration and invasion is well known. In addition, both factors are key components in cancer cell-related signaling. However, little information is available for uPA and PAI-1-associated signaling pathways in primary cancers and corresponding lymph node metastases. The aim of this study was to compare the expression of uPA and PAI-1-associated signaling proteins in 52 primary breast cancers and corresponding metastases. Proteins were extracted from formalin-fixed paraffin-embedded tissue samples of the primary tumors and metastases. Protein lysates were subsequently analyzed by reverse phase protein array for the expression of members of the PI3K/AKT (FAK, GSK3-β, ILK, pGSK3-β, PI3K, and ROCK) and the MAPK pathways (pp38, pSTAT3, and p38). A solid correlation of uPA expression existed between primary tumors and metastases, whereas PAI-1 expression did not significantly correlate between them. The correlations of uPA and PAI-1 with signaling pathways found in primary tumors did not persist in metastases. Analysis of single molecules revealed that some correlated well between tumors and metastases (FAK, pGSK3-β, ILK, Met, PI3K, ROCK, uPA, p38, and pp38), whereas others did not (PAI-1 and GSK3-β). Whether the expression of a protein correlated between tumor and metastasis or not was independent of the pathway the protein is related to. These findings hint at a complete deregulation of uPA and PAI-1-related signaling in metastases, which might be the reason why uPA and PAI-1 reached clinical relevance only for lymph node-negative breast cancer tissues.
PMCID: PMC3323931  PMID: 22496926
10.  Decentral gene expression analysis for ER+/Her2− breast cancer: results of a proficiency testing program for the EndoPredict assay 
Virchows Archiv  2012;460(3):251-259.
Gene expression profiles provide important information about the biology of breast tumors and can be used to develop prognostic tests. However, the implementation of quantitative RNA-based testing in routine molecular pathology has not been accomplished, so far. The EndoPredict assay has recently been described as a quantitative RT-PCR-based multigene expression test to identify a subgroup of hormone–receptor-positive tumors that have an excellent prognosis with endocrine therapy only. To transfer this test from bench to bedside, it is essential to evaluate the test–performance in a multicenter setting in different molecular pathology laboratories. In this study, we have evaluated the EndoPredict (EP) assay in seven different molecular pathology laboratories in Germany, Austria, and Switzerland. A set of ten formalin-fixed paraffin-embedded tumors was tested in the different labs, and the variance and accuracy of the EndoPredict assays were determined using predefined reference values. Extraction of a sufficient amount of RNA and generation of a valid EP score was possible for all 70 study samples (100%). The EP scores measured by the individual participants showed an excellent correlation with the reference values, respectively, as reflected by Pearson correlation coefficients ranging from 0.987 to 0.999. The Pearson correlation coefficient of all values compared to the reference value was 0.994. All laboratories determined EP scores for all samples differing not more than 1.0 score units from the pre-defined references. All samples were assigned to the correct EP risk group, resulting in a sensitivity and specificity of 100%, a concordance of 100%, and a kappa of 1.0. Taken together, the EndoPredict test could be successfully implemented in all seven participating laboratories and is feasible for reliable decentralized assessment of gene expression in luminal breast cancer.
PMCID: PMC3306560  PMID: 22371223
Breast cancer; Prognosis; mRNA; Quality control
11.  Biomarker analysis of cetuximab plus oxaliplatin/leucovorin/5-fluorouracil in first-line metastatic gastric and oesophago-gastric junction cancer: results from a phase II trial of the Arbeitsgemeinschaft Internistische Onkologie (AIO) 
BMC Cancer  2011;11:509.
The activity of the epidermal growth factor receptor (EGFR)-directed monoclonal antibody cetuximab combined with oxaliplatin/leucovorin/5-fluorouracil (FUFOX) was assessed in first-line metastatic gastric and oesophago-gastric junction (OGJ) cancer in a prospective phase II study showing a promising objective tumour response rate of 65% and a low mutation frequency of KRAS (3%). The aim of the correlative tumour tissue studies was to investigate the relationship between EGFR gene copy numbers, activation of the EGFR pathway, expression and mutation of E-cadherin, V600E BRAF mutation and clinical outcome of patients with gastric and OGJ cancer treated with cetuximab combined with FUFOX.
Patients included in this correlative study (n = 39) were a subset of patients from the clinical phase II study. The association between EGFR gene copy number, activation of the EGFR pathway, abundance and mutation of E-cadherin which plays an important role in these disorders, BRAF mutation and clinical outcome of patients was studied. EGFR gene copy number was assessed by FISH. Expression of the phosphorylated forms of EGFR and its downstream effectors Akt and MAPK, in addition to E-cadherin was analysed by immunohistochemistry. The frequency of mutant V600E BRAF was evaluated by allele-specific PCR and the mutation profile of the E-cadherin gene CDH1 was examined by DHPLC followed by direct sequence analysis. Correlations with overall survival (OS), time to progression (TTP) and overall response rate (ORR) were assessed.
Our study showed a significant association between increased EGFR gene copy number (≥ 4.0) and OS in gastric and OGJ cancer, indicating the possibility that patients may be selected for treatment on a genetic basis. Furthermore, a significant correlation was shown between activated EGFR and shorter TTP and ORR, but not between activated EGFR and OS. No V600E BRAF mutations were identified. On the other hand, an interesting trend between high E-cadherin expression levels and better OS was observed and two CDH1 exon 9 missense mutations (A408V and D402H) were detected.
Our finding that increased EGFR gene copy numbers, activated EGFR and the E-cadherin status are potentially interesting biomarkers needs to be confirmed in larger randomized clinical trials.
Trial registration
Multicentre clinical study with the European Clinical Trials Database number 2004-004024-12.
PMCID: PMC3252322  PMID: 22152101
12.  B3 Lesions: Radiological Assessment and Multi-Disciplinary Aspects 
Breast Care  2010;5(4):209-217.
B3 lesions comprise different histopathological entities that are considered benign but ‘of unknown biological potential’. These entities may act as risk indicators (for both breasts) or as non-obligatory precursors of malignancy. Being diagnosed at percutaneous breast biopsy, an additional risk of underestimate exists. Imaging appearances, histopathological appearance and risk of associated malignancy are presented. B3 lesions of high risk, which thus should usually be excised, include atypical ductal hyperplasia (ADH), pleomorphic or necrotic type of lobular neoplasia (LIN 3), and papillary lesions with atypias. Intermediate risk may be associated with classic lobular carcinoma in situ (LIN 2) or flat epithelial atypia (FEA), and low risk with radial sclerosing lesions (RSLs) and papillary lesions without atypias. LIN 1 is mostly an incidental finding acting as risk indicator. Follow-up is adequate if the initial diagnostic problem is solved. According to international guidelines, risk and subsequent recommendations should be discussed for each individual patient, taking into account biological risk, representative sampling, lesion size, lesion extent, percentage of lesion removal, other individual risks, and the possibility of surveillance. With vacuum-assisted breast biopsy (VABB), surgery may be avoided for more of the small lesions at low risk. Further data collection and diligent evaluation may help to better assess the individual risk, to better adapt treatment recommendations and avoid overtreatment.
PMCID: PMC3346165  PMID: 22590440
B3 lesion; Biopsy; Underestimate; Atypical hyperplasia
13.  Tenascin-C Enhances Pancreatic Cancer Cell Growth and Motility and Affects Cell Adhesion through Activation of the Integrin Pathway 
PLoS ONE  2011;6(6):e21684.
Pancreatic cancer (PDAC) is characterized by an abundant fibrous tissue rich in Tenascin-C (TNC), a large ECM glycoprotein mainly synthesized by pancreatic stellate cells (PSCs). In human pancreatic tissues, TNC expression increases in the progression from low-grade precursor lesions to invasive cancer. Aim of this study was the functional characterization of the effects of TNC on biologic relevant properties of pancreatic cancer cells.
Proliferation, migration and adhesion assays were performed on pancreatic cancer cell lines treated with TNC or grown on a TNC-rich matrix. Stable transfectants expressing the large TNC splice variant were generated to test the effects of endogenous TNC. TNC-dependent integrin signaling was investigated by immunoblotting, immunofluorescence and pharmacological inhibition.
Endogenous TNC promoted pancreatic cancer cell growth and migration. A TNC-rich matrix also enhanced migration as well as the adhesion to the uncoated growth surface of poorly differentiated cell lines. In contrast, adhesion to fibronectin was significantly decreased in the presence of TNC. The effects of TNC on cell adhesion were paralleled by changes in the activation state of paxillin and Akt.
TNC affects proliferation, migration and adhesion of poorly differentiated pancreatic cancer cell lines and might therefore play a role in PDAC spreading and metastasis in vivo.
PMCID: PMC3126842  PMID: 21747918
14.  High number of CD45RO+ tumor infiltrating lymphocytes is an independent prognostic factor in non-metastasized (stage I-IIA) esophageal adenocarcinoma 
BMC Cancer  2010;10:608.
The validation of novel prognostic indicators is of greatest interest for the management of esophageal adenocarcinoma (Barrett's cancer), particularly for non-metastasized (stage I-IIA) disease. The prognostic role of tumor infiltrating T-lymphocytes (TILs) in Barrett's cancer has not been reported so far. Here we evaluated the impact of TILs on survival, recurrence, and metastasis in Barrett's cancer, particularly in stage I-IIA patients.
The levels of the adaptive immune markers CD3, CD8, and CD45RO were analyzed by immunohistochemistry and image analysis in tissue microarrays consisting of tumor tissues of 118 patients with primary resected Barrett's cancer. The findings were correlated with clinicopathological parameters including patient outcome.
In multivariate analysis, a low density of intratumoral CD45RO+ immune cells was an independent unfavorable factor for disease-free survival in stages I-IIA patients (P = 0.004, RR = 4.7, 95% CI = 1.6-13.5) as well in the entire cohort (P = 0.048, RR = 2.0, 95% CI = 1.0-4.0). High CD3+ and CD45RO+ levels were associated with prolonged disease-free survival and overall survival as well with low recurrence rates of disease (P = 0.005 and P = 0.018, respectively). In addition, low CD3+ levels were correlated with a higher frequency of lymph node metastasis (P = 0.025).
This study demonstrates that the density of CD45RO+ TILs is an independent prognostic factor in non-metastasized (stage I-IIA) Barrett's cancer patients and indicates an important role for the adaptive immunologic microenvironment. The inclusion of CD45RO+ density may help to improve the management of stage I-IIA Barrett's cancer.
PMCID: PMC2988756  PMID: 21054833
15.  Prognostic significance of p21WAF1/CIP1, p27Kip1, p53 and E‐cadherin expression in gastric cancer 
Journal of Clinical Pathology  2007;60(7):756-761.
Gastric carcinoma is characterised by numerous genetic and epigenetic alterations that influence cell cycle progression, apoptosis and DNA repair. These alterations include down‐regulation of the cyclin‐dependent kinase (CDK) inhibitors p21WAF1/CIP1 and p27Kip1, and mutations of the tumour suppressor protein p53 and the cell adhesion molecule E‐cadherin. Combined evaluation of the prognostic significance of these alterations has not been reported in Mexican Mestizo patients.
To evaluate p21WAF1/CIP1, p27Kip1, p53 and E‐cadherin protein expression, including mutant E‐cadherin variants with deletion of exon 8 (del 8) or 9 (del 9), in gastric cancer from Mexican patients.
Immunohistochemistry for the above‐mentioned markers, including mutation‐specific E‐cadherin antibodies, was carried out in 69 gastric carcinomas; expression levels were correlated with histotype, tumour stage and prognosis.
Expression of p21WAF1/CIP1 alone or in combination with p27Kip1 or in the absence of p53 was associated with favourable prognosis. Staining of del 8 and del 9 E‐cadherin was found exclusively in patients negative for p53 and positive for p21WAF1/CIP1, suggesting that the p21WAF1/CIP1 regulatory function of p53 was intact.
Combined evaluation of the prognostic significance of cell cycle regulators and E‐cadherin should be performed. Even though patients negative for p53 and positive for p21WAF1/CIP1 have a favourable prognosis, it may have a negative influence on prognosis if they acquire in addition E‐cadherin mutations which have been shown previously to be associated with poor survival.
PMCID: PMC1995786  PMID: 17483253
p21WAF1/CIP1 ; p27Kip1 ; p53; E‐cadherin; gastric cancer
16.  Characterization of a naturally-occurring p27 mutation predisposing to multiple endocrine tumors 
Molecular Cancer  2010;9:116.
p27Kip1 (p27) is an important negative regulator of the cell cycle and a putative tumor suppressor. The finding that a spontaneous germline frameshift mutation in Cdkn1b (encoding p27) causes the MENX multiple endocrine neoplasia syndrome in the rat provided the first evidence that Cdkn1b is a tumor susceptibility gene for endocrine tumors. Noteworthy, germline p27 mutations were also identified in human patients presenting with endocrine tumors. At present, it is not clear which features of p27 are crucial for this tissue-specific tumor predisposition in both rats and humans. It was shown that the MENX-associated Cdkn1b mutation causes reduced expression of the encoded protein, but the molecular mechanisms are unknown. To better understand the role of p27 in tumor predisposition and to characterize the MENX animal model at the molecular level, a prerequisite for future preclinical studies, we set out to assess the functional properties of the MENX-associated p27 mutant protein (named p27fs177) in vitro and in vivo.
In vitro, p27fs177 retains some properties of the wild-type p27 (p27wt) protein: it localizes to the nucleus; it interacts with cyclin-dependent kinases and, to lower extent, with cyclins. In contrast to p27wt, p27fs177 is highly unstable and rapidly degraded in every phase of the cell-cycle, including quiescence. It is in part degraded by Skp2-dependent proteasomal proteolysis, similarly to p27wt. Photobleaching studies showed reduced motility of p27fs177 in the nucleus compared to p27wt, suggesting that in this compartment p27fs177 is part of a multi-protein complex, likely together with the degradation machinery. Studies of primary rat newborn fibroblasts (RNF) established from normal and MENX-affected littermates confirmed the rapid degradation of p27fs177 in vivo which can be rescued by Bortezomib (proteasome inhibitor drug). Overexpression of the negative regulators microRNA-221/222 plays no role in regulating the amount of p27fs177 in RNFs and rat tissues.
Our findings show that reduced p27 levels, not newly acquired properties, trigger tumor formation in rats, similarly to what has been observed in mice. The molecular characteristics of p27fs177 establish MENX as a useful preclinical model to evaluate compounds that inhibit p27 degradation for their efficacy against endocrine tumors.
PMCID: PMC2881881  PMID: 20492666
17.  The role of the pathologist in tissue banking: European Consensus Expert Group Report 
Virchows Archiv  2010;456(4):449-454.
Human tissue biobanking encompasses a wide range of activities and study designs and is critical for application of a wide range of new technologies (-“omics”) to the discovery of molecular patterns of disease and for implementation of novel biomarkers into clinical trials. Pathology is the cornerstone of hospital-based tissue biobanking. Pathologists not only provide essential information identifying the specimen but also make decisions on what should be biobanked, making sure that the timing of all operations is consistent with both the requirements of clinical diagnosis and the optimal preservation of biological products. This document summarizes the conclusions of a Pathology Expert Group Meeting within the European Biological and Biomolecular Research Infrastructure (BBMRI) Program. These recommendations are aimed at providing guidance for pathologists as well as for institutions hosting biobanks on how to better integrate and support pathological activities within the framework of biobanks that fulfill international standards.
PMCID: PMC2852521  PMID: 20157825
Pathology; Biobanks; Biomarkers; Harmonization; Standards; Translational research
18.  Activation of epidermal growth factor receptor results in Snail protein but not mRNA overexpression in endometrial cancer 
Reduced E-cadherin expression is associated with tumour progression of many carcinomas, including endometrial cancers. The transcription factor Snail is known as one of the most prominent transcriptional E-cadherin repressors; its regulation in cancer tissues, however, still remains unclear. Here, we report that activation of epidermal growth factor receptor (EGFR) resulted in overexpression of Snail and also identified critical downstream signalling molecules. Stimulation of two endometrial carcinoma cell lines with epidermal growth factor (EGF) lead to an increase of Snail protein expression. In primary human endometrioid endometrial carcinomas Snail protein expression correlated with the activated, phosphorylated form of EGFR (Tyr1086) as revealed by profiling 24 different signalling proteins using protein lysate microarrays. In addition, we observed an inverse correlation between Snail and E-cadherin protein levels in these tumours. Most likely, p38 MAPK, PAK1, AKT, ERK1/2 and GSK-3β are involved in the up-regulation of Snail downstream of EGFR. Snail mRNA expression did not show a correlation with activated EGFR in these tumours. Taken together, profiling of signalling proteins in primary human tissues provided strong evidence that EGFR signalling is involved in Snail protein overexpression.
PMCID: PMC4516533  PMID: 19604315
EGFR; Snail; E-cadherin; protein microarray; endometrial carcinoma; Her2
19.  Efficient shRNA delivery into B and T lymphoma cells using lentiviral vector-mediated transfer 
Journal of Hematopathology  2008;2(1):9-19.
RNA interference is a powerful tool for the functional analysis of proteins by specific gene knockdown. In this study, we devised a rapid and efficient way to screen suitable siRNA sequences and subsequently employ them for specific gene knockdown in usually hard-to-transfect lymphoid cell lines, using a self-inactivating lentiviral vector. Two proteins with different half-lives were chosen, cyclin D1 and STAT3. A specific lacZ reporter fusion assay was used to identify highly effective siRNA sequences. Only siRNA molecules with more than 85% of knockdown efficiency were selected for the generation of lentiviral transfer vectors. Transduction rates of 75–99% were achieved in the lymphoma cell lines Granta 519 (mantle cell lymphoma), Karpas 299, and SUDHL-1 (anaplastic large T cell lymphoma), as demonstrated by green fluorescent protein expression in fluorescence-activated cell sorting analysis. The high level of transduction efficiency allows RNA interference studies to be performed on transduced cells without further manipulation, such as cell sorting or cloning. The LacZ reporter system together with the lentivirus technology is a very important tool in the hematology field, which enables experiments in lymphoid cells that were not possible before.
Electronic supplementary material
The online version of this article (doi:10.1007/s12308-008-0020-x) contains supplementary material, which is available to authorized users.
PMCID: PMC2713496  PMID: 19669218
RNA interference; Efficient siRNA; β-galactosidase assay; Lentivirus transduction; Mantle cell lymphoma (MCL); Anaplastic large cell lymphoma (ALCL)
20.  MALDI imaging mass spectrometry for direct tissue analysis: a new frontier for molecular histology 
Histochemistry and Cell Biology  2008;130(3):421-434.
Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is a powerful tool for investigating the distribution of proteins and small molecules within biological systems through the in situ analysis of tissue sections. MALDI-IMS can determine the distribution of hundreds of unknown compounds in a single measurement and enables the acquisition of cellular expression profiles while maintaining the cellular and molecular integrity. In recent years, a great many advances in the practice of imaging mass spectrometry have taken place, making the technique more sensitive, robust, and ultimately useful. In this review, we focus on the current state of the art of MALDI-IMS, describe basic technological developments for MALDI-IMS of animal and human tissues, and discuss some recent applications in basic research and in clinical settings.
PMCID: PMC2522327  PMID: 18618129
MALDI imaging mass spectrometry; Tissue; In situ-proteomics; Pathology
21.  Genetic Alterations in Presumptive Precursor Lesions of Breast Carcinomas 
The hypothetical multistep model of breast carcinogenesis suggests a transition from normal epithelium to invasive carcinoma via intraductal hyperplasia (without and with atypia) and in situcarcinoma. These presumptive precursor lesions are currently defined by their histological features, and their prognosis is imprecisely estimated from indirect epidemiological evidence. Cytogenetic and molecular‐genetic analysis of these lesions give evidence for an accumulation of various genetic alterations during breast tumorigenesis. Using immuno‐histochemistry overexpression of the c‐erbB‐2 oncogene was found in ductal carcinoma in situ(DCIS), but not in atypical intraductal hyperplasia (AIDH) and intraductal hyperplasia (IDH). An expression of mutant p53 tumor suppressor gene as well as expression of cyclin D1 was identified in DCIS. In IDH lesions loss of heterozygosity (LOH) at various loci could be identified, and comparative genomic hybridization (CGH) and fluorescence in situhybridization (FISH) studies delivered evidence for DNA amplification on chromosomal region 20q13 in the early stage of IDH. However, little is currently known about genetic alterations in those premalignant lesions, and the chronology of genetic alterations and histopathological changes during carcinogenesis is mainly undiscovered. Figure 1 can be viewed in colour on‐23/aubele.htm
PMCID: PMC4618575  PMID: 12446956
22.  Chromosomal Imbalances are Associated with Metastasis-Free Survival in Breast Cancer Patients 
Multiple chromosomal imbalances have been identified in breast cancer using comparative genomic hybridization (CGH). Their association with the primary tumors' potential for building distant metastases is unknown. In this study we have investigated 39 invasive breast carcinomas with a mean follow‐up period of 99 months (max. 193 months) by CGH to determine the prognostic value of chromosomal gains and losses. The mean number of chromosomal imbalances per tumor was 6.5±0.7 (range 2 to 18). The most frequent alterations identified in more than 1/3 of cases were gains on chromosomes 11q13, 12q24, 16, 17, and 20q, and losses on 2q and 13q. A significantly different frequency of chromosomal aberrations (p≤0.05) was found between DNA‐diploid and non‐diploid tumors (gain on chromosome 17). Differences were also noted between tumors progressing to distant metastases within the period of follow‐up and those which do not (gains on 11q13 and 12q24; loss on 12q). Significant univariate correlations (p≤0.05) with the metastasis‐free survival of patients were found for lymph node status, the cytometrical determined DNA ploidy (diploid/non‐diploid) and anisokaryosis, and for DNA gains on 11q13, 12q24, 17, and 18p. An unexpected inverse correlation was found between clinical outcome and gains on 11q13 and 12q24. In multivariate analysis independent prognostic value, in addition to lymph node status, was found for chromosomal gains on 11q13, 12q24, 17 and 18p. Amplification on 20q, which did not correlate with metastasis‐free survival in a univariate analysis, showed weak prognostic significance in combination with the nodal status. The prognostic value of chromosomal alterations – some of them by inverse correlation – suggests an interaction and/or compensation of the involved amplified genes and their effects on the occurrence of distant metastases in breast cancer patients.
PMCID: PMC4618589  PMID: 12446957
CGH; prognosis; breast cancer; chromosomal imbalances; metastasis‐free survival
23.  Evaluation of C-ErbB-2 Overexpression and Her-2/neu Gene Copy Number Heterogeneity in Barrett’s Adenocarcinoma 
Amplifiction of the Her-2/neu gene is accompanied by overexpression of its cell surface receptor product, c‐erbB‐2 protein. To investigate the degree of intratumoural heterogeneity we applied immunohistochemistry in primary Barrett’s adenocarcinoma (BCA) (n = 6) and dysplasia adjacent to the carcinoma (n = 4). In addition, fluorescence in situ hybridisation (FISH) was performed in primary BCA (n=5) and dysplastic areas (n=4). For an objective evaluation digital image analysis and laser scanning microscopy were used. Five of six BCA showed a marked intratumoural heterogeneous staining pattern ranging from areas in which the tumour cells were negative or faintly positive to tumour areas with a strong staining of the entire membrane. Among the two dysplastic areas also a heterogenous staining pattern was observed. FISH analysis revealed marked heterogeneity of intratumoural gene copy number changes in all BCA showing populations with different fractions of cells with polysomy, low level amplification and high level amplification. One dysplasia showed a minor population with Her‐2/neu signal clusters. In conclusion, we observed marked intratumoural heterogeneity of c‐erbB‐2 protein overexpression and Her‐2/neu gene copy number in the majority of the primary BCA analyzed. Digital image analysis and laser scanning microscopy were helpful in quantifying the variations in protein expression and DNA copy number in individual tumour cells. The observed heterogeneity could hamper the exact diagnostic determination of the c‐erbB‐2 status in small biopsies and possibly influence the effectiveness of a potential c‐erbB‐2 targeting therapy.
Figures on‐1/walch.htm.
PMCID: PMC4615972  PMID: 11007435
Intratumoural heterogeneity; Her‐2/neu; c‐erbB‐2; fluorescence in situ hybridization; immunohistochemistry; laser scanning microscopy; digital image analysis; Barrett’s adenocarcinoma; dysplasia
24.  Heterogeneous Chromosomal Aberrations in Intraductal Breast Lesions Adjacent to Invasive Carcinoma 
There is evidence that breast cancer is a heterogeneous disease phenotypically as well as molecular biologically. So far, heterogeneity on the molecular biological level has not been investigated in potential precursor lesions, such as ductal hyperplasia (DH) and ductal carcinoma in situ (DCIS). In this study we applied comparative genomic hybridization (CGH) to formalin‐fixed, paraffin‐embedded breast tissue with DH and DCIS, adjacent to invasive ductal carcinoma (IDC), to screen these potential precursor lesions for whole genomic chromosomal imbalances. Laser‐microdissection was used to select pure cell populations from the sections. Isolated DNA was amplified by degenerate oligonucleotide primed PCR (DOP‐PCR) and further processed for CGH analysis.
Investigating multiple samples (n=25) from four patients we found an average of 5.6 ± 0.9 (mean ± SEM) chromosomal imbalances already present in DH. In the twelve DCIS lesions an average of 10.8 (±0.9) aberrations was identified with 14.8 (±0.8) aberrations in the four adjacent IDC lesions. The increasing number of chromosomal changes in parallel with the histopathological sequence corroborate the hypothesis, that the carcinomas may have developed through a sequential progression from normal to proliferative epithelium and eventually into carcinoma. However, heterogeneous results were identified in the multiple samples per entity from the same patient, demonstrated mainly in the DCIS samples in the chromosomal regions 6p, 9p, 11q, 16p and 17q, in the DH samples by 3p, 16p and 17q. This heterogeneous findings were most pronounced within the DH and was less in the DCIS and IDC samples. The only aberration consistently found in all samples – even in all DH samples – was amplification of the 20q13 region.
Our results demonstrate, that the applied combination of laser‐microdissection, DOP‐PCR and CGH, may serve to analyse breast carcinogenesis pathways in suitable histological material. However, so far, it is unclear how to handle heterogeneous results and these make identification of relevant changes more difficult. Setting a threshold and valuating only those chromosomal changes which are present in a majority of samples may be one possibility. This involves however, the risk that infrequent but possibly significant aberrations may be missed.
Figures on‐1/aubele.htm.
PMCID: PMC4617804  PMID: 11007434
25.  Diagnosis and Prognosis of Neuroendocrine Tumours of the Lung by Means of High Resolution Image Analysis 
Neuroendocrine tumours (NET) of the lung are divided in subtypes with different malignant potential. The first is the benign or low‐grade malignant tumours, well‐differentiated, called typical carcinoids (TC) and the second is the high‐grade malignant tumours, poorly differentiated of small (SCLC) or large cell type (LCLC). Between these tumour types lies the well‐differentiated carcinoma with a lower grade of malignancy (WDNEC). In clinical routine it is very important with regard to prognosis to distinguish patients with low malignant potential from those with higher ones. In this study 32 cases of SCLC, 13 of WDNEC and 14 of TC with a follow‐up time up to 7 years were collected. Sections 4 μm thick from paraffin embedded tissue were Feulgen stained. By means of high resolution image analysis 100 nuclei per case were randomly gathered to extract morphometric, densitometric and textural quantitative features. To investigate the ploidy status of the tumour the corrected DNA distribution was calculated. Stepwise linear discriminant analysis to differentiate the classes and Cox regression analysis for the survival time analysis were applied. Using chromatin textural and morphometric features in two two‐class discriminations, 11 of the 14 TC cases and 8 of the 13 WDNEC cases were correctly classified and 11/13 WDNEC cases and 28/32 SCLC cases, respectively. The WDNEC cases are more similar in chromatin structure to TC than to SCLC. For the survival analysis, only chromatin features were selected to differentiate patients with better and worse prognosis independent of staging and tumour type.
PMCID: PMC4617518  PMID: 10468408
Neuroendocrine lung tumours; carcinoid; small cell carcinoma; textural features; image analysis; DNA distribution; prognosis

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