Niels Grabe

Publications (50) View all

• Article: HPV-related methylation signature predicts survival in oropharyngeal squamous cell carcinomas.

  Efterpi Kostareli, Dana Holzinger, Olga Bogatyrova, Thomas Hielscher, Gunnar Wichmann, Michaela Keck, Bernd Lahrmann, Niels Grabe, Christa Flechtenmacher, Christopher R Schmidt, [......], Gerhard Dyckhoff, Andreas Dietz, Daniela Höfler, Michael Pawlita, Axel Benner, Franz X Bosch, Peter Plinkert, Christoph Plass, Dieter Weichenhan, Jochen Hess
  [show abstract] [hide abstract]
  ABSTRACT: High-risk types of human papilloma virus (HPV) are increasingly associated with oropharyngeal squamous cell carcinoma (OPSCC). Strikingly, patients with HPV-positive OPSCC are highly curable with ionizing radiation and have better survival compared with HPV-negative patients, but the underlying molecular mechanisms remain poorly understood. We applied an array-based approach to monitor global changes in CpG island hypermethylation between HPV-negative and HPV-positive OPSCCs and identified a specific pattern of differentially methylated regions that critically depends on the presence of viral transcripts. HPV-related alterations were confirmed for the majority of candidate gene promoters by mass spectrometric, quantitative methylation analysis. There was a significant inverse correlation between promoter hypermethylation of ALDH1A2, OSR2, GATA4, GRIA4, and IRX4 and transcript levels. Interestingly, Kaplan-Meier analysis revealed that a combined promoter methylation pattern of low methylation levels in ALDH1A2 and OSR2 promoters and high methylation levels in GATA4, GRIA4, and IRX4 promoters was significantly correlated with improved survival in 3 independent patient cohorts. ALDH1A2 protein levels, determined by immunohistochemistry on tissue microarrays, confirmed the association with clinical outcome. In summary, our study highlights specific alterations in global gene promoter methylation in HPV-driven OPSCCs and identifies a signature that predicts the clinical outcome in OPSCCs.
  The Journal of clinical investigation 05/2013; · 15.39 Impact Factor
• Article: Sequence mutations of the substrate binding pocket of stem cell factor and multidrug resistance protein ABCG2 in renal cell cancer: A possible link to treatment resistance.

  Inka Zoernig, Claudia Ziegelmeier, Bernd Lahrmann, Niels Grabe, Dirk Jäger, Niels Halama
  [show abstract] [hide abstract]
  ABSTRACT: ABCG2 is a multidrug cellular transport protein that is associated with resistance to certain treatments in patients, particularly anticancer treatment. The tumor-protective properties of ABCG2 expression are reported to be a feature of a subset of stem cell-like tumor cells. While protection against chemotherapy has been well analyzed, the role of ABCG2 in the treatment with tyrosine kinase inhibitors is only partially understood. Tyrosine kinase inhibitors are currently the main treatment option in irresectable renal cell carcinomas. To investigate possible underlying sequence variations in the ABCG2 gene with relevance to the functional properties of the protein, 36 patient samples were analyzed. Using sequence analysis and single-nucleotide polymorphism databases, sequence variations in the highly conserved domains of the binding pocket of ABCG2 were analyzed. The resulting variations were used for computational protein prediction algorithms to identify conformational alterations. A relevant shift from A to G at position 1376 (resulting in Y→C at 459 aa) was identified and found to be present in 8.3% of the patients. These patients are currently in follow-up after resection, thus, further analysis will reveal whether this mutation has relevance to treatment efficacy.
  Oncology Reports 03/2013; · 1.84 Impact Factor
• Article: Identification of oropharyngeal squamous cell carcinomas with active HPV16 involvement by immunohistochemical analysis of the retinoblastoma protein pathway.

  Dana Holzinger, Christa Flechtenmacher, Nataly Henfling, Ines Kaden, Niels Grabe, Bernd Lahrmann, Markus Schmitt, Jochen Hess, Michael Pawlita, Franz X Bosch
  [show abstract] [hide abstract]
  ABSTRACT: Viral oncogene RNA expression is regarded as reliable biomarker to identify oropharyngeal squamous cell carcinomas (OPSCC) with active HPV16 involvement. This study addressed whether the expression profile of the cellular proteins p16INK4a , pRb, Cyclin D1 and p53 provide surrogate marker combinations that identify OPSCC with active HPV16 in situations where only formalin-fixed biopsies are available. Protein expression was analyzed by immunohistochemistry on tissue microarrays created from 188 OPSCC of which the HPV16 DNA and RNA status had been established previously from snap-frozen biopsies. Associations of single markers and of marker combinations with HPV16 DNA, viral RNA expression patterns and overall survival as primary end point were evaluated by statistical analysis. Most tumors with active HPV16 involvement (RNA+ group; n=40) showed a specific protein pattern, i.e. high p16INK4a (80%), low pRb (85%), low Cyclin D1 (95%) and normal p53 (73%). This pattern was significantly different from the pattern observed in HPV DNA-negative tumors (HPV- group) and in HPV16 DNA-positive tumors lacking viral RNA expression patterns (RNA- group). The combination of high p16INK4a and low pRb levels was distinctly superior to p16INK4a alone; it was strongly associated with RNA+ tumors (OR 41.4, 95%CI 10.7-162.5), with improved survival (HR 0.37, 95%CI 0.2-0.8) and had best predictive values (78% sensitivity, 93% specificity, 78% PPV, 93% NPV). In conclusion, if only formalin-fixed biopsy material is available, the marker combination high p16INK4a /low pRb is well suited to identify OPSCC with biologically active HPV16 which represent a distinct OPSCC entity with improved prognosis. © 2013 Wiley Periodicals, Inc.
  International Journal of Cancer 03/2013; · 5.44 Impact Factor
• Article: Semantic focusing allows fully automated single-layer slide scanning of cervical cytology slides.

  Bernd Lahrmann, Nektarios A Valous, Urs Eisenmann, Nicolas Wentzensen, Niels Grabe
  [show abstract] [hide abstract]
  ABSTRACT: Liquid-based cytology (LBC) in conjunction with Whole-Slide Imaging (WSI) enables the objective and sensitive and quantitative evaluation of biomarkers in cytology. However, the complex three-dimensional distribution of cells on LBC slides requires manual focusing, long scanning-times, and multi-layer scanning. Here, we present a solution that overcomes these limitations in two steps: first, we make sure that focus points are only set on cells. Secondly, we check the total slide focus quality. From a first analysis we detected that superficial dust can be separated from the cell layer (thin layer of cells on the glass slide) itself. Then we analyzed 2,295 individual focus points from 51 LBC slides stained for p16 and Ki67. Using the number of edges in a focus point image, specific color values and size-inclusion filters, focus points detecting cells could be distinguished from focus points on artifacts (accuracy 98.6%). Sharpness as total focus quality of a virtual LBC slide is computed from 5 sharpness features. We trained a multi-parameter SVM classifier on 1,600 images. On an independent validation set of 3,232 cell images we achieved an accuracy of 94.8% for classifying images as focused. Our results show that single-layer scanning of LBC slides is possible and how it can be achieved. We assembled focus point analysis and sharpness classification into a fully automatic, iterative workflow, free of user intervention, which performs repetitive slide scanning as necessary. On 400 LBC slides we achieved a scanning-time of 13.9±10.1 min with 29.1±15.5 focus points. In summary, the integration of semantic focus information into whole-slide imaging allows automatic high-quality imaging of LBC slides and subsequent biomarker analysis.
  PLoS ONE 01/2013; 8(4):e61441. · 4.09 Impact Factor
• Article: Multistage histopathological image segmentation of Iba1-stained murine microglias in a focal ischemia model: Methodological workflow and expert validation.

  Nektarios A Valous, Bernd Lahrmann, Wei Zhou, Roland Veltkamp, Niels Grabe
  [show abstract] [hide abstract]
  ABSTRACT: A multistage workflow was developed for segmenting and counting murine microglias from histopathological brightfield images, in a permanent focal cerebral ischemia model. Automated counts are useful, since for the assessment of inflammatory mechanisms in ischemic stroke there is a need to quantify the brain's responses to post-ischemia, which primarily is the rapid activation of microglial cells. Permanent middle cerebral artery occlusion was induced in murine brain tissue samples. Positive cells were quantified by immunohistochemistry for the ionized calcium-binding adaptor molecule-1 (Iba1) as the microglia marker. Microglia cells were segmented in seven sequential steps: i) contrast boosting using quaternion operations, ii) intensity outlier normalization, iii) nonlocal total variation denoising, iv) histogram specification and contrast stretching, v) homomorphic filtering, vi) global thresholding, and vii) morphological filtering. Workflow counts were validated on an image subset, with ground-truth data acquired from manual counts conducted by a neuropathologist. Automated workflow matched ground-truth counts pretty well; 80 to 90% accuracy was achieved, as regards to time after pMCAO and correspondence to ischemic/non-ischemic tissue.
  Journal of neuroscience methods 12/2012; · 2.30 Impact Factor