Standard 2D histology of breast cancer

When breast cancer is diagnosed, the breast is usually examined and palpated by a doctor, after which the breast is imaged using mammography and, if necessary, with ultrasound. In addition, a large-core needle biopsy is collected from the tumour tissue.

The cancer type is classified by a histological analysis of the sample, by determining which cell type the cancer cells resemble the most. The most common breast cancer types are ductal and lobular carcinoma, the former meaning cancer that is developed in the cells of the milk ducts and the latter meaning cancer that begins in the lobules that produce milk. The invasive ductal carcinoma is the most common breast cancer since 70–80 % of invasive breast cancers are that type. (Heikkilä & Kärjä 2019)

When cancer cells are detected from a histological sample, a cancer cell is usually dif-ferent than normal cell in size or in shape. Cancer cells also vary from each other within the sample. In addition to the appearance of cells, changes at the level of cell nucleus can also be noticed. A cancerous nucleus is usually bigger in size and darker than normal cell nucleus because of bigger amount of DNA, and this can be also noticed when typical staining methods are in use. As cells of the breast tissue normally form ducts and lobules, cancer cells can be noticed to form deformed structures or clusters of cells that are not organized at all. (American Cancer Society, 2015)

As the histological type of cancer has been determined, the grade of the cancer is also classified by a Bloom–Richardson system which grades the cancer based on three cri-teria: the amount of tissue that forms tubular structure, the mitotic count and the pleo-morphism of the cell nuclei. Each of the categories is scored from 1 to 3 points, and the overall score determines the tumour grade from 1 to 3, the lowest indicating nearly nor-mal tissue and the highest the most abnornor-mal and most poorly differentiated tissue. The first criterion, the tubular structure, is usually found in normal breast tissue since the cells form ducts and therefore such tissue gets 1 point from that category, whereas cancer cells do not form that much of such organized structures, thus earning higher points. The

mitotic count is determined as the amount of dividing cells in certain field of view which appears to be higher in cancerous tissue, resulting in higher points. The last category, the nuclear pleomorphism, covers examination of cell nuclei that differ from normal nuclei in size or shape. For instance, irregular large nuclei or higher amount of nucleoli within the nuclei leads to higher points. (He et al. 2011; Heikkilä & Kärjä 2019) Figure 6 shows an example of H&E stained sample of grade 1 ductal carcinoma. The tumour can be seen as purple area on bottom right of the figure since ductal carcinoma usually appears as solid groups or cords of tumour cells. Adipose tissue (in the middle), which is also called fat tissue, forms the majority of the breast volume and stroma (top right) is a part of the tissue which has a connective role. (The human protein atlas)

Figure 6. H&E stained histology slice of ductal breast carcinoma in which the tu-mour can be seen on bottom right. (The human protein atlas)

In addition to the cancer grade, the stage of the cancer is also determined before treat-ment. It contains defining the tumour location and size using TNM-classification where T refers to primary tumour, N to regional lymph nodes and M to distant metastases. Sim-plified, the idea is to measure the tumour size and determine if the cancer cells have spread to regional lymph nodes or other parts of the body. To perform TNM-classifica-tion, histological analysis is needed. In addiTNM-classifica-tion, the histological types of breast cancer, such as ductal and lobular carcinoma, can be furthermore divided into different molecular and genetical subtypes based on gene profile analysis. However, since the gene profile analysis is not very common in clinical use, a rough estimation can be performed by immunohistochemical staining of oestrogen receptors, progesterone receptors and hu-man epidermal growth factor 2 (HER2) which is an oncogene. Pathologist reports the

results as the proportion of positively stained nuclei, and a proper treatment method can be chosen based on the receptor status. Normal breast tissue has receptors for oestro-gen and progesterone, and some breast cancer cells have also one or both of these receptors which makes hormone therapy treatment possible in which the hormone levels are lowered or drugs are used to stop a certain hormone from affecting the cancer cells.

Similarly, some breast cancer cells have too many HER2-receptors on their cell mem-brane which promote the growth of the cancer cells. There are various very effective targeted treatments available for HER2-positive cancers. HER2-status can be either de-termined by immunohistochemistry or by fluorescence in situ hybridization of the HER2-genes that may be present with too many copies. (Heikkilä & Kärjä 2019)

The earliest signs of breast cancer are small calcium deposits, calcifications, within the breast tissue. While they are very common and usually not malignant, it is important to be able to detect and distinguish suspicious calcifications from normal cases since they can also indicate invasive cancer types. They are usually noticed on a mammogram as white dots and, based on their size, calcifications are divided into macro- and microcal-cifications. Most of the detected calcifications are diagnosed as ductal carcinoma in situ (DCIS) which is the earliest form of breast cancer and not invasive. However, Mordang et al. (2018) studied the sensitivity of mammogram in calcification detection and the sen-sitivity was noticed to be only 45,5 % which could be improved. They stated that the sensitivity should be improved since the current stage of it leads to 68,4 % of the cancer-related calcifications to be diagnosed not until the cancer has developed into invasive stage. This could be achieved, for instance, by combining some other imaging technique with mammogram or by developing a specific method that could detect early signs of cancer with higher sensitivity while keeping the amount of false positives at current level.

Table 1 aggregates all of the histological methods that are in use when breast cancer is diagnosed.

General analysis

- variation in cell size or shape - variation in nucleus size or shape - variation in arrangement of cells

Cancer type Origin: in which cell type the cancer is developing

Cancer grade

Bloom–Richardson system - tubular structures - mitotic count

- pleomorphism of the cell nuclei

➔ Scoring from 1 to 3

Cancer stage

TNM-classification - tumour size

- regional lymph node involvement - presence of metastatic spread Molecular subtype /

Calcifications Presence of micro- and macrocalcifications and their malignancy

Next, the possibilities of 3D histology methods for detection of breast cancer are studied and analysed.