# Deep Learning within a DICOM WSI Viewer for Histopathology
## 2 Materials and Methods
### Datasets  
Images from various hospitals and diagnostic tissues must be used to demonstrate the system's compatibility. To accomplish this, a dataset composed of 153 cases of invasive and in situ ductal breast cancer carcinomas was used. There were 54 incisional biopsiesand 99 excisional biopsies. The biopsies and WSIs were prepared and acquired by four centers, Nottingham University Hospital (NHS Trust), Vilniaus Universiteto Ligonine Santaros Klinikos (VULSK), Servicio de Salud de Castilla La Mancha (SESCAM) and Servicio Andaluz de Salud (SAS), as part of the AIDPATH project [35]. All specimens were stained with hematoxylin and eosin (H&E), human epidermal growth factor receptor 2 (HER2) and Ki-67 antigen-specific monoclonal antibody (Ki67) using standard procedures. Breast cancer was chosen as a use case due to its high prevalence. It was also reasonable to include samples of the three stains chosen for this study due to their importance in histopathological diagnosis and prognosis. One of the most common tissue stains used in histology is the H&E stain. It is the most commonly used stain in medical diagnosis and is frequently used as the gold standard. Pathologists usually stain histological sections of suspected cancer biopsies with H&E. H&E is an abbreviation for hematoxylin and eosin, two histological stains. Cell nuclei are stained a purplish blue by hematoxylin, and the extracellular matrix and cytoplasm are stained pink by eosin, with other structures taking on different hues and color intensities. Thus, a pathologist can easily differentiate between a cell's nuclear and cytoplasmic components, and the overall color of the stain reveals the tissue sample's cell arrangement and distribution. In addition, immunohistochemical (IHC) staining is commonly used in the diagnosis of abnormal cells, such as those found in cancerous tumors. Specific molecular markers are associated with specific cellular events, such as proliferation or apoptosis. The HER2 and Ki67 IHC markers, in particular, are extremely important in cancer prognosis. HER2 oncogene overexpression has been linked to the development and progression of certain aggressive types of breast cancer. In recent years, HER2 has emerged as a critical biomarker and therapeutic target for approximately 30% of breast cancer patients. Patients with HER2 amplification in breast cancer have a more aggressive form of the disease, as well as greater resistance to conventional treatments. Ki67, on the other hand, is a cellular marker for proliferation. Thus, Ki67 is an excellent marker for determining the growth fraction of a cell population. Ki67 identifies a high proliferative subset of patients with estrogen receptor-positive breast cancer who can benefit more from adjuvant chemotherapy. The percentage of Ki67-positive tumor cells (the Ki67 labeling index) is frequently linked to cancer progression. All the WSIs were acquired at 20\(\times\) and 40\(\times\) magnification. The digital scanning was performed using a Hamamatsu NanoZoomer C9600 (TIF format) for the NHS Trust and a Leica DM-4000 (SVS format) for SESCAM and SAS. During standard and quality-measured routines, pathologists from the NHS Trust, VULSK, SESCAM and SAS provided the ground truth. At least two specialists reviewed and labeled each case. All the images had an average resolution of 90,000 \(\times\) 50,000 pixels and 1 GB file size in a proprietary format. From the HER2 IHC test, a score of 0 to 3+ was assigned to each sample depending on the amount of HER2 protein stained. A score of 0 to 1+ is considered as HER2-negative. A score of 2+ is regarded as being on the borderline. A 3+ score is defined as HER2-positive. In addition, hotspot localization and quantification of IHC Ki67-positive staining are crucial. The aggressiveness of a breast tumor is indicated by a high proportion of Ki67-positive staining in a biopsy, which also suggests a higher proliferation rate of the tumor.