56P - Quantification BCL6 tissue expression using FISH and immunohistochemistry in diffuse large B-cell lymphoma by digital image analysis

Abstract

Background: Computational method was developed to study how nucleus alterations would influence on cell cycle progression in cancer. It was verified that information retrieved from chromatin organisation areas and their heterogeneity can be used to infer nuclear morphological features. Diffuse large B-cell lymphoma (DBLC) is heterogeneous entity due to its morphologic alteration of cells. Numerous studies have attempted to further understand how nuclear morphology and expression antiapototic genes interfere on cell interaction and pathogenesis. Objective: To determine the immunohistochemical (bcl-6) molecular marker and morphological characteristics of nucleus (FISH probe BCL6) using digital pathology algorithm in patients with DBLC. Methods: The study included data on 14 patients with diffuse large-cell B-cell lymphoma (mean age 50 ± 15 years) before therapy. TMA templates were prepared for BCL6 FISH break apart probe as well as for immunohistochemical (ihc) marker bcl-6. Fluorescence microscopy images obtained from tissue slides by histologic scanner. The number of stained pixels / area was quantified using computer quantitative ihc, nuclear and histopattern algorithms for each TMA core. The study of the nuclear morphology was pursued through the analysis of the first and second sets of DAPI staining images and the study of distribution green and red signals into cells. In this work, a semi-automatic bioinformatic application was implemented to manage the images sets and process them. Results: Concerning the nuclear morphological features evaluated (area, density of chromatin - eccentricity and solidity), the results demonstrated that the quantification of the nuclei area was the most meaningful parameter to disclose significant biological information as translocation of BCL6 gene. Besides it was associated with aneuplody and amplification of BCL6 genes and overexpression of bcl-6 protein. These nuclear morphological features revealed significant differences between cells with translocation of BCL6 and cells without translocations. Conclusions: These findings support the hypothesis that morphology of nuclei are mediated by the rearrangement of the chromatin density and cytoskeleton interaction. Legal entity responsible for the study: Anna Artemyeva. Funding: Petrov Cancer Research Center. Disclosure: All authors have declared no conflicts of interest.