Semi-Automatic Quantification Of mRNA Expression In Whole-Slide Tissue Images

INTRODUCTION / BACKGROUND: For complex diseases, commercially available anti- bodies directed again specific protein sometimes lack the specificity required for clinical purpose. Therefore, novel techniques have been proposed recently to better assess the expression status of molecules at the mRNA level, e.g. by in situ hybridization. With such a technique, single-molecule signals can then be quantified on a cell-by-cell basis in whole tissue slides. However, highly heterogeneous expression of mRNA is often observed at the tissue level, requiring the development of accurate and reproducible quantification methods. AIMS: In this work, our aim is to propose and evaluate a methodology for the quantification of mRNA expression in digital slides to help biomedical researchers to analyze their large-scale imaging data. In particular, we will work on a dataset of breast cancer tissue samples and evaluate the accuracy of such a methodology to quantify Breast cancer susceptibility gene 1 (BRCA1) mRNA expression levels. BRCA1 is a tumor suppressor gene associated with the triple negative breast cancer (TNBC) subtype. An accurate technique to determine BRCA1 protein tumoral expression status in TNBC would allow for informed decision and choosing adapted treatments. METHODS: Data: To design and evaluate our methodology, we use the data from [1]. In that study, the BRCA1 mRNA expression was assessed by in situ hybridization using RNAscope technology [2] (ACD-Bioke) for formalin-fixed, paraffin-embedded tissue samples. 88 glass slides were scanned (Hamamatsu, 40X, 0.23µm/pixel) and transferred on a Cytomine server (http://www.cytomine.be/) [3]. These slides correspond to 62 tumours (TNBC subtype). Using Cytomine web manual annotation tools, a total of more than 200 regions of interest were manually drawn by an expert in these whole-slide images. Then, independent assessment was performed by two observers in these ROI: they manually draw point annotations corresponding to tens of thousands of BRCA1 mRNA expression signals. ALGORITHm: We have developed the Icytomine plugin to communicate between Icy and Cytomine. It allows the importation of the subtumor images at a desired resolution and the expert annotations from Cytomine and the exportation of detection results from Icy software (http://icy.bioimageanalysis.org) [4]. In order to quantify mRNA signals, we combined several image processing routines in the Icy. Our workflow is composed of three steps: 1) color deconvolution [5] to extract the dark brown staining, 2) detection of spots based on undecimated wavelet transform [6], 3) post-processing to remove artifacts based on size, shape and color features. This transform is fast and ideal for representing isotropic objects. We will use a colocalisation coefficient to quantify the concordance between the experts. For a purpose of automation, we use scripts and protocols of Icy. RESULTS: An illustration of our results is given in Figure Our ongoing large-scale empirical study stresses the need for more confident ground-truths hence more robust algorithms. Interestingly, our current strategy detects accurately spot locations. We believe it will help us to create a larger ground-truth dataset using expert proofreading. We will report at the conference our best quantitative results. Overall, we believe that our approach will speedup in a confident way the quantification of mRNA signals in whole-slide tissue samples

Detection of wheat allergens using 2D Western blot and mass spectrometry

peer reviewedBackground: Wheat allergy is relatively common and the associated clinical manifestations depend on the involved molecular allergens as well as on the way of exposure. Different symptoms have been described: wheat-dependent exercise-induced anaphylaxis (WDEIA), atopic dermatitis (AD) and pollen rhinitis (PR). Traditional diagnostic methods do not allow accurate molecular identification of the allergens that are essential for risk assessment and for the choice of the most adapted treatment. Methods: Standardized total protein extracts obtained from wheat seeds were separated by 2D electrophoresis. Twenty-one sera with high wheat-specific immunoglobulin E (sIgE) levels were classified into three patients groups based on their clinical profile. These sera were tested by Western blot on 2D separated standardized wheat protein extract and their sIgE sensitization profiles were compared. Results: Specific sensitization profiles were identified for each phenotype group. For WDEIA, protein spots around 37 kDa (pH 6-9) and 37–50 kDa (pH 5-6) were identified. For AD, spots were observed around 50 kDa (pH 9), 10 kDa (pH 9) and 20 to 75 kDa (pH3). For PR, specific spots were located around 90 kDa (pH 9). The mass spectrometry (UHPLC-MS/MS) analysis of these identified spots pointed out several potential interesting allergens: Tri a 26, Tri a bA, Tri a 34, Tri a tritin. Conclusions: The present study allowed the identification of different protein areas specific to these studied groups. The protein spots of interest were identified by UHPLC-MS/MS. It has been possible to establish a link between a specific symptomatology and the newly identified responsible allergens. © 2019 Elsevier B.V

BRCA1 germline mutation and glioblastoma development: report of cases

Background Germline mutations in breast cancer susceptibility gene 1 (BRCA1) increase the risk of breast and ovarian cancers. However, no association between BRCA1 germline mutation and glioblastoma malignancy has ever been highlighted. Here we report two cases of BRCA1 mutated patients who developed a glioblastoma (GBM). Cases presentation Two patients diagnosed with triple negative breast cancer (TNBC) were screened for BRCA1 germline mutation. They both carried a pathogenic mutation introducing a premature STOP codon in the exon 11 of the BRCA1 gene. Few years later, both patients developed a glioblastoma and a second breast cancer. In an attempt to clarify the role played by a mutated BRCA1 allele in the GBM development, we investigated the BRCA1 mRNA and protein expression in breast and glioblastoma tumours for both patients. The promoter methylation status of this gene was also tested by methylation specific PCR as BRCA1 expression is also known to be lost by this mechanism in some sporadic breast cancers. Conclusion Our data show that BRCA1 expression is maintained in glioblastoma at the protein and the mRNA levels, suggesting that loss of heterozygosity (LOH) did not occur in these cases. The protein expression is tenfold higher in the glioblastoma of patient 1 than in her first breast carcinoma, and twice higher in patient 2. In agreement with the high protein expression level in the GBM, BRCA1 promoter methylation was not observed in these tumours. In these two cases, despite of a BRCA1 pathogenic germline mutation, the tumour-suppressor protein expression is maintained in GBM, suggesting that the BRCA1 mutation is not instrumental for the GBM development