Classifying breast cancer and fibroadenoma tissue biopsies from paraffined stain-free slides by fractal biomarkers in Fourier Ptychographic Microscopy

Breast cancer is one of the most spread and monitored pathologies in high-income countries. After breast biopsy, histological tissue is stored in paraffin, sectioned and mounted. Conventional inspection of tissue slides under benchtop light microscopes involves paraffin removal and staining, typically with H&E. Then, expert pathologists are called to judge the stained slides. However, paraffin removal and staining are operator-dependent, time and resources consuming processes that can generate ambiguities due to non-uniform staining. Here we propose a novel method that can work directly on paraffined stain-free slides. The method is automatic, independent from the operator, and provides classification of different portions of the tissue image with very high accuracy. Besides, it returns a guide map to help pathologist to judge the different tissue portions based on the likelihood these can be associated to a breast cancer or fibroadenoma biomarker. We use Fourier Ptychography as a quantitative phase-contrast microscopy method, which allows accessing a very wide field of view (i.e., square millimeters) in one single image while guaranteeing at the same time high lateral resolution (i.e., 0.5 microns). This imaging method is multi-scale, since it enables looking at the big picture, i.e. the complex tissue structure and connections, with the possibility to zoom-in up to the single cell level. In order to handle this informative image content, we introduce elements of fractal geometry as a multi-scale analysis method. We show the effectiveness of fractal features in describing fibroadenoma and breast cancer from six patients with very high accuracy. The proposed method could significantly simplify the steps of tissue analysis and make it independent from the sample preparation, the skills of the lab operator and the pathologist. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was supported by project POR CIRO (Campania Imaging for Research in Oncology) funded by Regione Campania (Italy). This work was partially supported by the Italian Ministry of Health ('Ricerca Corrente' project). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The study was approved by the Ethics Committee of IRCCS Pascale (Naples, Italy) with reference number 3/19 approved on 29 May 2019. All methods were performed in compliance with standard operating procedures and in accordance with the Declaration of Helsinki and each patient participated in the study by signing written informed consent. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors