Wednesday, 17 January 2018

Quantitative immunohistochemical analysis

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Olivier DEBEIR - Christine DECAESTECKER - Xavier MOLES LOPEZ - Yves-Rémi VAN EYCKE

Project description

Example of immunohistochemical (IHC) stained slide

The quantification of immunostaining by means of digital image analysis allows the characterization of protein expression revealed by immunohistochemistry (IHC) on normal or pathological tissue slices. In contrast to other methods of evaluation of protein expression, this approach enables morphological controls and detailed tissue and cell localization to be carried out, avoiding the problems due to cell and tissue heterogeneity. This constitutes a promising approach for the identification of tissue-based biomarkers useful for diagnostic, prognostic and/or therapeutic purposes.

This project is is supported by the F.N.R.S. (Belgian National Fund for Scientific Research) and carried out in close collaboration with the Pathology Department of the Erasme Hospital (Prof. I. Salmon). It is thus centered on problems commonly encountered by pathologists in their research activities and aims to provide original and pragmatic solutions based on an engineering approach.

IHC biomarker processing workflow

It consists in:

  • Standardization of the image acquisition process
  • Automated methods for whole slide imaging (WSI) quality assessment (including image normalization process)
  • Robust morphological and IHC feature extraction
  • Multiresolution registration of serial slide images
  • IHC staining colocalization measurement
  • Data base construction (including clinical features) and integrated data analysis
  • Identification and validation of biomarkers by means of multivariate data analysis and machine learning


Diapath platform

Resulting from our close collaboration with the Pathology Department of the Erasme Hospital, DIAPATH (Digital Image Analysis in Pathology) is a unit of the Center for Microscopy and Molecular Imaging (CMMI). This center is a biomedical imaging facility supported by the "Région Wallonne" and the European Union ("FEDER-Convergence" funds). The DIAPATH unit aspires to become a leader in histological image analysis services for academic and industrial partners.



We developed a tool able to help pathologists in the task of characterizing the proliferative activity on high-grade gliomas. Ki67 is a common biomarker used in clinical practice to assess the proliferative activity of tumors. Faced with large images, pathologists require tools that can help them identify tumor regions that exhibit high proliferating activity, called "hot-spots" (HSs). The new tool is capable of helping pathologists to identify Ki67 HSs in whole tumor section images that are produced by slide scanners. We showed that this tool strongly improves the consistency among pathologists in this task of HS identification.

In order to compare the performance and the robustness of different clustering algorithms, we produce a set of 50 different datasets. These datasets are publicly available on the page CLUSTERING METHODS APPLIED IN THE DETECTION OF KI67 HOT-SPOTS

Representative publications


L. Verset, J. Tommelein, X. Moles Lopez, C. Decaestecker, T. Boterberg, E. De Vlieghere, I. Salmon, M. Mareel, M. Bracke, O. De Wever, P. Demetter,
Impact of neoadjuvant therapy on cancer-associated fibroblasts in rectal cancer.
Radiotherapy and oncology, Vol. 116, 3, pp. 449-54, 2015
Bibtex : info:hdl:2013/200672
Note : SCOPUS: cp.j
Abstract : Cancer-associated fibroblasts (CAFs) are increasingly recognised as promoters of tumour progression. It is poorly investigated whether cancer management protocols, such as neoadjuvant radio(chemo)therapy, have an impact on CAFs and, by consequence, on tumour progression. This prompted us to study the impact of neoadjuvant radio(chemo)therapy on the α-SMA/epithelial area ratio in rectal cancer, and the impact of this ratio on recurrence-free survival.

L. De Smedt, J. Lemahieu, S. Palmans, O. Govaere, T. Tousseyn, E. van Cutsem, H. Prenen, S. Tejpar, M. Spaepen, G. Matthijs, C. Decaestecker, X. Moles Lopez, P. Demetter, I. Salmon, X. Sagaert,
Microsatellite instable vs stable colon carcinomas: analysis of tumour heterogeneity, inflammation and angiogenesis.
British Journal of Cancer, Vol. 113, 3, pp. 500-9, 2015
Bibtex : info:hdl:2013/205313
Note : SCOPUS: ar.j
Abstract : Microsatellite instability (MSI) accounts for 15\% of all colorectal tumours. Several specific clinicopathologicals (e.g., preference for the proximal colon over the distal colon, improved prognosis and altered response to chemotherapeutics) are described for this subset of tumours. This study aimed to analyse morphological, inflammatory and angiogenic features of MSI vs microsatellite stable (MSS) tumours.

T. Gevaert, X. Moles Lopez, X. Sagaert, L. Libbrecht, T. Roskams, S. Rorive, C. Decaestecker, I. Salmon, D. De Ridder,
Morphometric and quantitative immunohistochemical analysis of disease-related changes in the upper (suburothelial) lamina propria of the human bladder dome.
PloS one, Vol. 10, 5, pp. e0127020, 2015
Bibtex : info:hdl:2013/200671
Note : SCOPUS: ar.j
Abstract : The upper (suburothelial) lamina propria (ULP) is a distinct region in the human bladder with dense populations of interstitial cells (IC), fine vascular networks and variable development of muscularis mucosae (MM). It is more and more obvious that the ULP plays an important role in bladder physiology and bladder disease, and in the present study we have quantified changes in the cellular key players of the ULP in bladders from patients with carcinoma in situ (CIS), multiple sclerosis (MS) and bladder pain syndrome (BPS). Tissue samples for the different patient groups were obtained from radical cystectomy-specimens. Standardized immunohistochemistry with a panel of specific cell markers was used to characterise the ULP cellular structures, followed by digitalised morphometry and quantitative staining analysis. Alterations in the ULP area were most pronounced in MS bladders, but also present in BPS and CIS bladders. We observed an increased thickness and increased variability in thickness of the ULP IC area in MS and BPS bladders; a significantly increased development of MM in MS bladders; a changed organization of vascular plexuses in the lamina propria in most pathologic bladders and a changed phenotype of ULP IC: a significantly decreased expression of progesterone receptor in MS bladders and a trend towards decreased expression of alpha-smooth muscle actin in BPS bladders. We show here for the first time the presence of disease-specific changes in organisation and/or phenotype of the different key players of the ULP area in human bladder. The present findings further support the hypothesis that the ULP area is involved and altered in different bladder diseases.

X. Moles Lopez, P. Barbot, Y. Van Eycke, L. Verset, A. Trepant, L. Larbanoix, I. Salmon, C. Decaestecker,
Registration of whole immunohistochemical slide images: an efficient way to characterize biomarker colocalization.
Journal of the American Medical Informatics Association, Vol. 22, 1, pp. 86-99, 2015
Bibtex : info:hdl:2013/177042
Abstract : Extracting accurate information from complex biological processes involved in diseases, such as cancers, requires the simultaneous targeting of multiple proteins and locating their respective expression in tissue samples. This information can be collected by imaging and registering adjacent sections from the same tissue sample and stained by immunohistochemistry (IHC). Registration accuracy should be on the scale of a few cells to enable protein colocalization to be assessed.

J. Allard, X. Moles Lopez, K. Li, O. Blanchard, P. Barbot, S. Rorive, C. Decaestecker, R. Pochet, D. Bohl, A. Lepore, I. Salmon, C. Nicaise,
Immunohistochemical toolkit for tracking and quantifying xenotransplanted human stem cells.
Regenerative medicine, Vol. 9, 4, pp. 437-452, 2014
Bibtex : info:hdl:2013/174921
Note : Journal Article
Abstract : Biomarker-based tracking of human stem cells xenotransplanted into animal models is crucial for studying their fate in the field of cell therapy or tumor xenografting.

X. Moles Lopez, E. D'Andrea, P. Barbot, A. Bridoux, S. Rorive, I. Salmon, O. Debeir, C. Decaestecker,
An Automated Blur Detection Method for Histological Whole Slide Imaging
PloS one, Vol. 8, 12, 2013
Bibtex : info:hdl:2013/152899
Note : SCOPUS: ar.j
Abstract : Whole slide scanners are novel devices that enable high-resolution imaging of an entire histological slide. Furthermore, the imaging is achieved in only a few minutes, which enables image rendering of large-scale studies involving multiple immunohistochemistry biomarkers. Although whole slide imaging has improved considerably, locally poor focusing causes blurred regions of the image. These artifacts may strongly affect the quality of subsequent analyses, making a slide review process mandatory. This tedious and time-consuming task requires the scanner operator to carefully assess the virtual slide and to manually select new focus points. We propose a statistical learning method that provides early image quality feedback and automatically identifies regions of the image that require additional focus points.

L. Verset, J. Tommelein, X. Moles Lopez, C. Decaestecker, M. Mareel, M. Bracke, I. Salmon, O. De Wever, P. Demetter,
Epithelial expression of FHL2 is negatively associated with metastasis-free and overall survival in colorectal cancer
British Journal of Cancer, Vol. 109, 1, pp. 114-120, 2013
Bibtex : info:hdl:2013/149047
Note : SCOPUS: ar.j
Abstract : Background:Four-and-a-half LIM domains protein 2 (FHL2) is a component of the focal adhesion structures and has been suggested to have a role in cancer progression. It has been shown to be overexpressed in the colorectal cancer (CRC).Methods:Here, we examined a possible prognostic value of FHL2 in CRC. Immunohistochemistry for FHL2 was performed on 296 CRCs without distant metastases at the time of surgery. Staining in the epithelial compartment was quantitatively evaluated using image analysis, and results were related to clinical variables. Antibody specificity was tested using small-interfering RNA transfection in hTERT-immortalised myofibroblasts.Results:Varying degrees of cytoplasmic FHL2 expression by neoplastic epithelial cells were detectable in all cases. Higher FHL2 expression in the epithelial compartment was an independent adverse prognostic factor. Multivariate Cox analysis shows that expression in the tumour invasion front (P<0.001) as well as in the centre of the tumour (P<0.001) was associated with metachronous metastases independently of the clinicopathological variables; expression in the tumour invasion front was also associated with overall survival independently of the clinicopathological variables (P<0.01).Conclusion:Higher FHL2 expression is involved in CRC progression and correlates with the development of metachronous metastases and overall survival, suggesting that FHL2 is an independent adverse prognostic indicator for CRC. © 2013 Cancer Research UK. All rights reserved.

M. Le Mercier, D. Hastir, X. Moles Lopez, N. De Nève, C. Maris, A. Trepant, S. Rorive, C. Decaestecker, I. Salmon,
A simplified approach for the molecular classification of glioblastomas.
PloS one, Vol. 7, 9, 2012
Bibtex : info:hdl:2013/132976
Note : Journal Article
Abstract : Glioblastoma (GBM) is the most common malignant primary brain tumors in adults and exhibit striking aggressiveness. Although GBM constitute a single histological entity, they exhibit considerable variability in biological behavior, resulting in significant differences in terms of prognosis and response to treatment. In an attempt to better understand the biology of GBM, many groups have performed high-scale profiling studies based on gene or protein expression. These studies have revealed the existence of several GBM subtypes. Although there remains to be a clear consensus, two to four major subtypes have been identified. Interestingly, these different subtypes are associated with both differential prognoses and responses to therapy. In the present study, we investigated an alternative immunohistochemistry (IHC)-based approach to achieve a molecular classification for GBM. For this purpose, a cohort of 100 surgical GBM samples was retrospectively evaluated by immunohistochemical analysis of EGFR, PDGFRA and p53. The quantitative analysis of these immunostainings allowed us to identify the following two GBM subtypes: the "Classical-like" (CL) subtype, characterized by EGFR-positive and p53- and PDGFRA-negative staining and the "Proneural-like" (PNL) subtype, characterized by p53- and/or PDGFRA-positive staining. This classification represents an independent prognostic factor in terms of overall survival compared to age, extent of resection and adjuvant treatment, with a significantly longer survival associated with the PNL subtype. Moreover, these two GBM subtypes exhibited different responses to chemotherapy. The addition of temozolomide to conventional radiotherapy significantly improved the survival of patients belonging to the CL subtype, but it did not affect the survival of patients belonging to the PNL subtype. We have thus shown that it is possible to differentiate between different clinically relevant subtypes of GBM by using IHC-based profiling, a method that is advantageous in its ease of daily implementation and in large-scale clinical application.

L. Ferdinande, C. Decaestecker, L. Verset, A. Mathieu, X. Moles Lopez, A. Negulescu, T. Van Maerken, I. Salmon, C. Cuvelier, P. Demetter,
Clinicopathological significance of indoleamine 2,3-dioxygenase 1 expression in colorectal cancer.
British Journal of Cancer, Vol. 106, 1, pp. 141-147, 2012
Bibtex : info:hdl:2013/106373
Abstract : Background:Indoleamine 2,3-dioxygenase 1 (IDO1) is a tryptophan-catabolising enzyme that induces immune tolerance by modulating T-cell responses. Carcinomas may create an immunosuppressive state via IDO1 expression. Here we examined a possible contribution of IDO1 on this phenomenon and investigated whether IDO1 has prognostic value in colorectal cancer (CRC).Methods:IDO1 expression was investigated by quantitative PCR and western blotting in three colon cancer cell lines, in basal state and after interferon (IFN)-γ stimulation. Semi-quantitative immunohistochemistry was used to evaluate IDO1 expression in 265 pT1-4N0-2Mx-staged CRCs. Results were related to clinical variables and correlated with amounts of CD3(+) and CD8(+) T lymphocytes, which were quantitatively evaluated using image analysis.Results:In vitro expression of IDO1 depended on IFN-γ stimulation. Higher IDO1 expression at the tumour invasion front was an independent adverse prognostic factor in pT1-4N1Mx-staged CRC. It was associated with overall survival (P=0.001) and with metachronous metastases (P=0.018). IDO1 expression was not associated with the presence of CD3(+) or CD8(+) T lymphocytes.Conclusion:Higher IDO1 expression at the tumour invasion front is involved in CRC progression and correlates with impaired clinical outcome, suggesting that IDO1 is an independent prognostic indicator for CRC.British Journal of Cancer advance online publication, 22 November 2011; doi:10.1038/bjc.2011.513

X. Moles Lopez, O. Debeir, C. Maris, S. Rorive, I. Roland, M. Saerens, I. Salmon, C. Decaestecker,
Clustering methods applied in the detection of Ki67 hot-spots in whole tumor slide images: An efficient way to characterize heterogeneous tissue-based biomarkers.
Cytometry. Part A, Vol. 81, 9, pp. 765-775, 2012
Bibtex : info:hdl:2013/127791
Note : Journal Article
Abstract : Whole-slide scanners allow the digitization of an entire histological slide at very high resolution. This new acquisition technique opens a wide range of possibilities for addressing challenging image analysis problems, including the identification of tissue-based biomarkers. In this study, we use whole-slide scanner technology for imaging the proliferating activity patterns in tumor slides based on Ki67 immunohistochemistry. Faced with large images, pathologists require tools that can help them identify tumor regions that exhibit high proliferating activity, called "hot-spots" (HSs). Pathologists need tools that can quantitatively characterize these HS patterns. To respond to this clinical need, the present study investigates various clustering methods with the aim of identifying Ki67 HSs in whole tumor slide images. This task requires a method capable of identifying an unknown number of clusters, which may be highly variable in terms of shape, size, and density. We developed a hybrid clustering method, referred to as Seedlink. Compared to manual HS selections by three pathologists, we show that Seedlink provides an efficient way of detecting Ki67 HSs and improves the agreement among pathologists when identifying HSs. © 2012 International Society for Advancement of Cytometry.

S. Rorive, X. Moles Lopez, C. Maris, A. Trepant, S. Sauvage, N. Sadeghi-Meibodi, I. Roland, C. Decaestecker, I. Salmon,
TIMP-4 and CD63: new prognostic biomarkers in human astrocytomas.
Modern pathology, Vol. 23, 10, pp. 1418-1428, 2010
Bibtex : info:hdl:2013/68379
Note : Journal Article
Abstract : Based on the molecular profiling of astrocytomas, we previously identified a series of genes involved in astrocytoma invasion. Of these, tissue inhibitor of metalloproteinase-4 (TIMP-4) was found to be overexpressed in pilocytic astrocytomas relative to diffuse astrocytomas of any histological grade. Although some data suggest that TIMP-4 may be an anti-tumoral actor in astrocytomas, recent findings challenge this concept. The present study aims to investigate the diagnostic and prognostic values of TIMP-4 and its putative partner CD63 in human astrocytomas. Tissue microarray and image analysis were first carried out to quantitatively analyze the immunohistochemical expression of these proteins in 471 gliomas including 354 astrocytomas. Pathological semi-quantitative scores of both markers' expression were then established and correlated to astrocytoma diagnosis and patient prognosis. TIMP-4 and CD63 expressions were both overexpressed in astrocytomas compared with oligodendrogliomas (P<0.001) and in pilocytic astrocytomas compared with grade II diffuse astrocytomas (P<0.001). In glioblastomas, high TIMP-4/CD63 co-expression scores were identified as independent prognostic factors associated with progression and shorter survival. In conclusion, this work provides the first evidence of a TIMP-4/CD63 association in astrocytoma tumor cells. It identifies TIMP-4 and CD63 as markers of the astrocytic phenotype in patients with gliomas. In addition, this work highlights the contribution of high TIMP-4/CD63 co-expression to the adverse outcomes of patients with glioblastomas.

C. Decaestecker, X. Moles Lopez, N. D'Haene, I. Roland, S. Guendouz, C. Duponchelle, A. Berton, O. Debeir, I. Salmon,
Requirements for the valid quantification of immunostains on tissue microarray materials using image analysis.
Proteomics, Vol. 9, 19, pp. 4478-4494, 2009
Bibtex : info:hdl:2013/52938
Note : Journal Article
Abstract : Antibody-based proteomics applied to tissue microarray (TMA) technology provides a very efficient means of visualizing and locating antigen expression in large collections of normal and pathological tissue samples. To characterize antigen expression on TMAs, the use of image analysis methods avoids the effects of human subjectivity evidenced in manual microscopical analysis. Thus, these methods have the potential to significantly enhance both precision and reproducibility. Although some commercial systems include tools for the quantitative evaluation of immunohistochemistry-stained images, there exists no clear agreement on best practices to allow for correct and reproducible quantification results. Our study focuses on practical aspects regarding (i) image acquisition (ii) segmentation of staining and counterstaining areas and (iii) extraction of quantitative features. We illustrate our findings using a commercial system to quantify different immunohistochemistry markers targeting proteins with different expression patterns (cytoplasmic, nuclear or membranous) in colon cancer or brain tumor TMAs. Our investigations led us to identify several steps that we consider essential for standardizing computer-assisted immunostaining quantification experiments. In addition, we propose a data normalization process based on reference materials to be able to compare measurements between studies involving different TMAs. In conclusion, we recommend certain critical prerequisites that commercial or in-house systems should satisfy in order to permit valid immunostaining quantification.


L. Verset, J. Tommelein, X. Moles Lopez, C. Decaestecker, T. Boterberg, E. De Vlieghere, I. Salmon, M. Mareel, M. Bracke, O. De Wever, P. Demetter,
Impact of neoadjuvant therapy on cancer-associated fibroblast in rectal cancer
Conference: Joint Meeting of the British Division of the International Academy of Pathology and the Pathological Society of Great Britain & Ireland(8th: 23 – 25 June 2015: Dublin), 2015
Bibtex : info:hdl:2013/223057
Note : Language of publication: en

L. De Smedt, J. Lemahieu, S. Palmans, O. Govaere, T. Tousseyn, E. van Cutsem, H. Prenen, S. Tejpar, C. Decaestecker, X. Moles Lopez, P. Demetter, I. Salmon, X. Sagaert,
Microsatellite instability versus microsatellite stability in colon carcinoma: documentation of tumour heterogeneity and inflammation
Conference: Belgian Week of Gastroenterology(XXVIIth : February 2015: Brussels), 2015
Bibtex : info:hdl:2013/223062
Note : Language of publication: en

Y. Van Eycke, X. Moles Lopez, J. Allard, M. Derock, S. Rorive, I. Salmon, C. Decaestecker,
Multiresolution registration of whole slide images to evidence and quantify virtual colocalization of tissue-based biomarkers
Conference: Digital Pathology Congress(4-5 December 2014: London), 2014
Bibtex : info:hdl:2013/193430
Note : Language of publication: en
Abstract : Extracting relevant information from actors involved in complex biological processes (such as cancers or treatment responses) requires to target different antigens simultaneously. Multichromogenic (brightfield) immunohistochemistry (IHC) suffers from limitations that notably prevent the analysis of proteins expressed in the same cellular compartment. We thus developed an alternative based on the analysis of adjacent, or serial, tissue sections on which different proteins were targeted by means of standard IHC. For this analysis, we developed a multiresolution method for registering serial slide images. This method uses the pyramidal and multimodal registration framework of the elastix software to optimize two parametric registrations. The first, low-resolution registration, is applied on the 1X equivalent magnification images (4,000 by 3,000 pixels). The result of this first step was then used to initialize high-resolution registrations independently performed on 20X equivalent fields of view. Our method shows accuracy levels compatible with biomarker colocalization characterization. Indeed, registration error on serial slides was evaluated to be at most between 20 µm and 80 µm, depending respectively of the presence or absence of histological structures in the tissue (e.g. in colonic tumor as compared to brain gliomas). In the latter situation, a sequential IHC technique applied on the same slide can be usefully employed. This “Sequential Immunoperoxidase Labeling and Erasing” (SIMPLE) method is based on cycles of staining/digitization/erasing, where after IHC staining and slide digitization, staining is erased through an antibody elution technique. We improved the original SIMPLE method and successfully utilized it to identify antigens expressed in the same cellular compartment of high-grade glioma samples. We tested our registration on the virtual slides so obtained and achieved very good results, i.e. about 5 µm of registration error. We then implemented a method to extract biomarker colocalization measurements taking the level of registration error into account and validated our complete procedure by comparison to colocalization information obtained by means of double staining (with different cell locations).

X. Moles Lopez, C. Maris, T. Chattaway, M. Remmelink, B. Weynand, S. Rorive, C. Decaestecker, I. Salmon,
SecundOS: A Belgian digital pathology initiative to implement an inter-university network for second opinions and collaboration between expert pathologists.
Conference: Digital Pathology Congress(4-5 December 2014: London), 2014
Bibtex : info:hdl:2013/193431
Note : Available at (Abstract No 11)

X. Moles Lopez, P. Barbot, N. D'Haene, C. Maris, S. Rorive, I. Salmon, C. Decaestecker,
How Digital Image Analysis Applied to Immunohistochemistry Helps To Assess Biomarker Coexpression.
Conference: 102nd USCAP Annual Meeting, 2013
Bibtex : info:hdl:2013/193241
Note : Language of publication: na

L. Verset, J. Tommelein, X. Moles Lopez, C. Decaestecker, M. Mareel, M. Bracke, I. Salmon, O. De Wever, P. Demetter,
Investigation of a possible link between epithelial four-and-a-half LIM domains protein 2 expression and prognosis in colorectal cancer
Conference: 2013 Gastrointestinal Cancers Symposium, 2013
Bibtex : info:hdl:2013/193428
Note : Language of publication: en

X. Moles Lopez, O. Debeir, C. Maris, I. Roland, I. Salmon, C. Decaestecker,
Ki-67 hot-spots detection on glioblastoma tissue sections
Proc. of the 2010 IEEE International Symposium on Biomedical Imaging: From Nano to Macro (ISBI 2010), pp. pp 149-152, Rotterdam, 2010
Bibtex : info:hdl:2013/70188
Note : Language of publication: en

Books chapters

Moles Lopez, Xavier, Debeir, Olivier, Salmon, Isabelle, Decaestecker, Christine,
Whole slide imaging and analysis for biomarker evaluation in digital pathology
A. Méndez-Vilas (Ed.), Microscopy: advances in scientific research and education, Vol. 2, pp. 776-787, 2014
Bibtex : info:hdl:2013/175598
Note : Language of publication: na