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Currently: industrial collaborator at the LISA
Previously: PhD student

ULB - LISA CP165/57
50, Av. F.Roosevelt
1050 Bruxelles

PhD Thesis Research

Development of methods and tools for image analysis and automatic feature extraction in brightfield tissue images (project page:Quantitative immunohistochemical analysis). The specific goal is to develop methods for immunohistochemical staining detection and characterization.

PhD Thesis ULB, 2014



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.

R. Marée, B. Stévens, L. Rollus, N. Rocks, X. Moles Lopez, I. Salmon, D. Cataldo, L. Wehenkel,
A rich internet application for remote visualization and collaborative annotation of digital slides in histology and cytology
Diagnostic Pathology, Vol. 8, Suppl 1, pp. S26, 2013
Bibtex : info:hdl:2013/176838
Note : Language of publication: en

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.

N. D'Haene, C. Maris, S. Rorive, X. Moles Lopez, J. Rostang, C. Marchessoux, L. Pantanowitz, A. Parwani, I. Salmon,
Comparison study of five different display modalities for whole slide images in surgical pathology and cytopathology in Europe
Progress in biomedical optics and imaging, Vol. 8676, 2013
Bibtex : info:hdl:2013/148928
Note : SCOPUS: cp.p
Abstract : User experience with viewing images in pathology is crucial for accurate interpretation and diagnosis. With digital pathology, images are being read on a display system, and this poses new types of questions: such as what is the difference in terms of pixelation, refresh lag or obscured features compared to an optical microscope. Is there a resultant change in user performance in terms of speed of slide review, perception of adequacy and quality or in diagnostic confidence? A prior psychophysical study was carried out comparing various display modalities on whole slide imaging (WSI) in pathology at the University of Pittsburgh Medical Center (UPMC) in the USA. This prior study compared professional and non-professional grade display modalities and highlighted the importance of using a medical grade display to view pathological digital images. This study was duplicated in Europe at the Department of Pathology in Erasme Hospital (Université Libre de Bruxelles (ULB)) in an attempt to corroborate these findings. Digital WSI with corresponding glass slides of 58 cases including surgical pathology and cytopathology slides of varying difficulty were employed. Similar non-professional and professional grade display modalities were compared to an optical microscope (Olympus BX51). Displays ranged from a laptop (DELL Latitude D620), to a consumer grade display (DELL E248WFPb), to two professional grade monitors (Eizo CG245W and Barco MDCC-6130). Three pathologists were selected from the Department of Pathology in Erasme Hospital (ULB) in Belgium to view and interpret the pathological images on these different displays. The results show that non-professional grade displays (laptop and consumer) have inferior user experience compared to professional grade monitors and the optical microscope. © 2013 SPIE.

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.

M. Cnop, M. Igoillo Esteve, M. Rai, A. Begu, Y. Serroukh, C. Depondt, A. Musuaya, I. Marhfour, L. Ladrière, X. Moles Lopez, D. Lefkaditis, F. Moore, J. Brion, M. Cooper, A. Schapira, A. Clark, A. Koeppen, P. Marchetti, M. Pandolfo, D. Eizirik, F. Fery,
Central role and mechanisms of beta-cell dysfunction and death in Friedreich ataxia- associated diabetes
Annals of neurology, Vol. 72, pp. 971-982, 2012
Bibtex : info:hdl:2013/136306
Note : Language of publication: en
Abstract : Abstract Objective: Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disease caused in almost all cases by homozygosity for a GAA trinucleotide repeat expansion in the frataxin gene. Frataxin is a mitochondrial protein involved in iron homeostasis. FRDA patients have a high prevalence of diabetes, the pathogenesis of which is not known. We aimed to evaluate the relative contribution of insulin resistance and β-cell failure and the pathogenic mechanisms involved in FRDA diabetes. Methods: Forty-one FRDA patients, 26 heterozygous carriers of a GAA expansion, and 53 controls underwent oral and intravenous glucose tolerance tests. β-Cell proportion was quantified in postmortem pancreas sections from 9 unrelated FRDA patients. Using an in vitro disease model, we studied how frataxin deficiency affects β-cell function and survival. Results: FRDA patients had increased abdominal fat and were insulin resistant. This was not compensated for by increased insulin secretion, resulting in a markedly reduced disposition index, indicative of pancreatic β-cell failure. Loss of glucose tolerance was driven by β-cell dysfunction, which correlated with abdominal fatness. In postmortem pancreas sections, pancreatic islets of FRDA patients had a lower β-cell content. RNA interference–mediated frataxin knockdown impaired glucose-stimulated insulin secretion and induced apoptosis in rat β cells and human islets. Frataxin deficiency sensitized β cells to oleate-induced and endoplasmic reticulum stress-induced apoptosis, which could be prevented by the incretins glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide. Interpretation: Pancreatic β-cell dysfunction is central to diabetes development in FRDA as a result of mitochondrial dysfunction and higher sensitivity to metabolic and endoplasmic reticulum stress-induced β-cell death. ANN NEUROL 2012;72:971–982

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.

I. Marhfour, X. Moles Lopez, D. Lefkaditis, I. Salmon, F. Allagnat, S. Richardson, N. Morgan, D. Eizirik,
Expression of endoplasmic reticulum stress markers in the islets of patients with type 1 diabetes.
Diabetologia, Vol. 55, 9, pp. 2417-2420, 2012
Bibtex : info:hdl:2013/135708
Note : Journal Article
Abstract : Endoplasmic reticulum (ER) stress may play a role in cytokine-mediated beta cell death in type 1 diabetes, but it remains controversial whether ER stress markers are present in islets from type 1 diabetic individuals. Therefore, we evaluated by immunostaining the expression of markers of the three main branches of the ER stress response in islets from 13 individuals with and 15 controls without type 1 diabetes (eight adults and seven children).

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

N. D'Haene, C. Koopmansch, J. Allard, F. Hulet, X. Moles Lopez, Y. Van Eycke, C. Decaestecker, I. Salmon,
The prognostic value of the VEGFR-1 and -2 combinations in endothelial cells of colorectal cancer.
Conference: Belgian Week of Pathology 2015 (October 2015: Ghent, Belgium), 2015
Bibtex : info:hdl:2013/224048
Note : abstract P01 (p. 156)

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


X. Moles Lopez,
Characterization and Colocalization of Tissue-Based Biomarker Expression by Quantitative Image Analysis: Development and Extraction of Novel Features
Bibtex : info:hdl:2013/209330
Note : Doctorat en Sciences de l'ingénieur
Abstract : Proteins are the actual actors in the (normal or disrupted) physiological processes and immunohistochemistry (IHC) is a very efficient mean of visualizing and locating protein expression in tissue samples. By comparing pathologic and normal tissue, IHC is thus able to evidence protein expression alterations. This is the reason why IHC plays a grow- ing role to evidence tissue-based biomarkers in clinical pathology for diagnosing var- ious diseases and directing personalized therapy. Therefore, IHC biomarker evaluation significantly impacts the adequacy of the therapeutic choices for patients with serious pathologies, such as cancer. However, this evaluation may be time-consuming and dif- ficult to apply in practice due to the absence of precise positive cut-off values as well as staining (i.e. protein expression) heterogeneity intra- and inter-samples. Quantifying IHC staining patterns has thus become a crucial need in histopathology. For this task, automated image analysis has multiple advantages, such as avoiding the evidenced ef- fects of human subjectivity. The recent introduction of whole-slide scanners opened a wide range of possibilities for addressing challenging image analysis problems, includ- ing the identification of tissue-based biomarkers. Whole-slide scanners are devices that are able to image whole tissue slides at resolutions up to 0.1 micrometers per pixels, often referred to as virtual slides. In addition to quantification of IHC staining patterns, virtual slides are invaluable tools for the implementation of digital pathology work- flows. The present work aims to make several contributions towards this current digital shift in pathology. Our first contribution was to propose an automated virtual slide sharpness assessment tool. Although modern whole-slide scanner devices resolve most image standardization problems, focusing errors are still likely to be observed, requiring a sharpness assessment procedure. Our proposed tool will ensure that images provided to subsequent pathologist examination and image analysis are correctly focused. Virtual slides also enable the characterization of biomarker expression heterogeneity. Our sec- ond contribution was to propose a method to characterize the distribution of densely stained regions in the case of nuclear IHC biomarkers, with a focus on the identification of highly proliferative tumor regions by analyzing Ki67-stained tissue slides. Finally, as a third contribution, we propose an efficient mean to register virtual slides in order to characterize biomarker colocalization on adjacent tissue slides. This latter contribution opens new prospects for the analysis of more complex questions at the tissue level and for finely characterizing disease processes and/or treatment responses./Les protéines sont les véritables acteurs des processus physiologiques (normaux ou per- turbés) et l’immunohistochimie (IHC) est un moyen efficace pour visualiser et localiser leur expression au sein d’échantillons histologiques. En comparant des échantillons de tissus pathologiques et normaux, l’IHC permet de révéler des altérations dans des pro- fils d’expression protéique. C’est pourquoi l’IHC joue un rôle de plus en plus important pour mettre en évidence des biomarqueurs histologiques intervenant dans le diagnos- tic de diverses pathologies et dans le choix de thérapies personnalisées. L’évaluation de l’expression de biomarqueurs révélés par IHC a donc des répercussions importantes sur l’adéquation des choix thérapeutiques pour les patients souffrant de pathologies graves, comme le cancer. Cependant, cette évaluation peut être chronophage et difficile à appliquer en pratique, d’une part, à cause de l’hétérogénéité de l’expression protéique intra- et inter-échantillon, d’autre part, du fait de l’absence de critères de positivité bien définis. Il est donc devenu crucial de quantifier les profils d’expression de marquages IHC en histopathologie. A cette fin, l’analyse d’image automatisée possède de multiples avantages, comme celui d’éviter les effets de la subjectivité humaine, déjà démontrés par ailleurs. L’apparition récente des numériseurs de lames histologiques complètes, ou scanners de lames, a permis l’émergence d’un large éventail de possibilités pour traiter des problèmes d’analyse d’image difficiles menant à l’identification de biomar- queurs histologiques. Les scanners de lames sont des dispositifs capables de numériser des lames histologiques à une résolution pouvant atteindre 0,1 micromètre par pixel, expliquant la dénomination de "lames virtuelles" des images ainsi acquises. En plus de permettre la quantification des marquages IHC, les lames virtuelles sont des outils indis- pensables pour la mise en place d’un flux de travail numérique en pathologie. Le travail présenté ici vise à fournir plusieurs contributions au récent changement de cap vers une numérisation de la discipline médicale qu’est l’anatomie pathologique. Notre première contribution consiste en un outil permettant d’évaluer automatiquement la netteté des lames virtuelles. En effet, bien que les scanners de lames résolvent la plupart des pro- blèmes liés à la standardisation de l’acquisition, les erreurs de focus restent fréquentes, ce qui nécessite la mise en place d’une procédure de vérification de la netteté. L’outil que nous proposons assurera la netteté des images fournies à l’examen du pathologiste et à l’analyse d’image. Les lames virtuelles permettent aussi de caractériser l’hétérogénéité de l’expression de biomarqueurs. Ainsi, la deuxième contribution de ce travail repose sur une méthode permettant de caractériser la distribution de régions densément marquées par des biomarqueurs IHC nucléaires. Pour ce travail, nous nous sommes concentrés sur l’identification de régions tumorales présentant une forte activité proliférative en analysant des lames virtuelles révélant l’expression de la protéine Ki67. Finalement, la troisième contribution de ce travail fut de proposer un moyen efficace de recaler des lames virtuelles dans le but de caractériser la colocalisation de biomarqueurs IHC révé- lés sur des coupes de tissu adjacentes. Cette dernière contribution ouvre de nouvelles perspectives pour l’analyse de questions complexes au niveau histologique ainsi que la caractérisation fine de processus pathologiques et de réponses thérapeutiques.


X. Moles Lopez,
Bibtex : info:hdl:2013/115482