The Kuwaiti population is a growing one, and so is the incidence of cancer among the Kuwaitis. In females, the most common types of malignancies include breast cancer, followed by thyroid cancer, cervical uteri and colorectal carcinomas, and ovarian cancer. Breast cancer accounts for 30.3% of all cancer types, and death occurs in approximately 43% of patients 1.

The amount of research addressing breast carcinoma in Kuwait is still minimal, and nation-wide studies are still lacking. Accordingly, little is known so far about the disease. Based on our clinical encounters with breast cancer patients at Mubarak al-Kabeer Hospital, which is one of the five major public hospitals in the State of Kuwait, we have noticed that these patients seem to be presenting at a relatively young age with the disease at an advanced stage. Therefore, we sought to conduct a comprehensive analysis of the pathobiological characteristics of the tumours in an attempt to provide the clinicians with a better picture about the biological behaviour of the tumours including any particular trend that could be present. This could hopefully help the clinicians better understand the disease manifestation in the Kuwaiti population, and, accordingly, help them develop a platform on which further disease investigation and clinical management strategies could be based.

In this study, we put much emphasis on carefully examining the pathobiological characteristics of the tumour. These characteristics included tumour margin, histological types and subtypes, tumour surrounding breast tissue type, tumour grade, mitotic index, tumour size, nuclear pleomorphism, stage of the disease, lymphocyte involvement, breast laterality, nature of surgical management (operation performed), and degree of expression of the prognostic markers Her-2, oestrogen receptor (ER), and progesterone receptor (PgR). We also investigated any possible significant association between each of the above characteristics and the degree of expression of Her-2, ER, and PgR.

One hundred and sixty-six breast cancer cases seen at Mubarak Al-Kabeer Hospital, were reviewed. All the pathology reports and the H&E slides pertaining to these cases were collected. We selected the cases where complete information regarding the patient and the tumour were available. The Mubarak Al-Kabeer Hospital provides annual health care to around 750,000 people living in the Mubarak Al-Kabeer, Hawally, Salmiya, and Jabriya districts. The study was approved by the Human Ethics Committee at the Faculty of Medicine, Health Science Centre, Kuwait University, and it conformed to the provisions of the Declaration of Helsinki.

For immunohistochemical staining, the following standard procedure was followed 2. Sections were cut at 4 μm thickness, mounted onto silane-coated slides (S21.1910.110, Novocastra, Newcastle upon Tyne, UK), and left to dry overnight at 37°C. They were then deparaffinized, re-hydrated, and underwent antigen retrieval (Epitope Retrieval PH6, RE7115, Novocastra) by microwaving (Daewoo KOR-161G, 1000 W, 2450 MHz, 10 power levels, Seoul, South Korea) for 20 minutes. After cooling down to room temperature, the sections were incubated for 15 minutes with 3% hydrogen peroxide to block any endogenous peroxidase activity, washed with TBS, and incubated with goat serum (NCL-G-SERUM, Novocastra) for 20 minutes to block any non-specific staining. They were then washed with TBS, and incubated with 200 μl of primary antibodies for 1 hour at 37°C in a humidified rotator. The dilution of the primary antibody against Her-2 (Clone 10A7, NCL-CBE-356, Novocastra) was 1:40, while those of ER (Clone 6F11, NCL-ER-6F11, Novocastra) and PgR (Clone 16, NCL-PgR-312, Novocastra) were 1:40 and 1:100, respectively. The sections were then washed with TBS, and incubated with the secondary link antibody (Novolink Max RE7280-K, Novocastra) for 30 minutes. This was followed by washing with TBS and incubation with the tertiary antibody (Novolink Max RE7280-K, Novocastra) for 30 minutes. Finally, the sections were washed with TBS and incubated for 10 minutes with DAB (Novolink Max RE7280-K, Novocastra) chromogen contrasted with hematoxylin counterstain. Positive and negative control slides were used in each staining experiment. The positive controls were breast carcinomas known to be positive for Her-2, ER, and PgR. The negative controls included sections of breast carcinomas known to be negative for Her-2, ER, and PgR, and sections taken from the same tissue block but incubated with antibody diluent instead of the primary antibody.

The scoring system developed by Allred et al. 2 in relation to ER, PgR, and Her-2 staining was followed. Accordingly, both the proportion and intensity of staining are taken into consideration. A proportion score indicated the proportion of positive tumour cells on the entire slide, and it ranged from 0 to 5. An intensity score indicated the average staining intensity of positive tumour cells, and it ranged from 0 to 3. Both scores were then added, to obtain a total score ranging from 0 to 8. This scoring system has been followed in numerous experiments and has resulted in an inter- and intra observer reproducibility of more than 90% 2. Based on this scoring system, any total score between 6 and 8 was considered overexpression in our study. If the total score was 0, the tumour was considered lacking expression.

The tumours were typed according to the WHO classification system 3. Although it is beyond the scope of this manuscript to provide details of such classification, we will summarize the major points. To start with, such classification includes invasive versus non-invasive breast carcinomas, based on the infiltration of the basal membrane by the tumour cells. The non-invasive carcinomas are classified as ductal carcinoma in situ (DCIS) or lobular carcinoma in situ (LCIS), based on the involvement of the ducts or the lobules forming the breast tissue. The DCIS tumours include the following subtypes based on the morphology: comedo, cribriform, solid, papillary, micropapillary, and apocrine. The invasive tumours are further classified as ductal-not otherwise specified, lobular, tubular/cribriform, colloid (mucinous), medullary, papillary, comedo, Paget's disease, adenoid, and apocrine.

The modified Bloom-Richardson-Elston histological system was used for grading the invasive carcinomas 4. This aims at examining the growth pattern as well as the biological characteristics of differentiation of these carcinomas. Accordingly, the following criteria were used:

1) Formation of the gland tubules and acini

a) characteristic formation of tubules (>75%) = 1 point

b) moderate formation of tubules (10–75%) = 2 points

c) little or without tubules at all (<10%) = 3 points

2) Pleomorphism of cancer cell nuclei (abnormalities in size, structure, and shape)

a) isomorphism of nuclei or small variability in shape, size, and structure of nuclei = 1 point

b) moderate variability in shape, size, and structure of nucleus = 2 points

c) marked characteristic polymorphism = 3 points

3) Mitosis per 10 high power fields

a) <10 mitosis = 1 point

b) 10–20 mitosis = 2 points

c) >20 mitosis = 3 points

By adding up the points of the above criteria, the level of tumour differentiation could then be calculated as follows: grade I (well differentiated; 3–5 points), grade II (moderately differentiated; 6–7 points), grade III (poorly differentiated; 8–9 points).

As far as staging the carcinomas is concerned, we followed the criteria set by the American Joint Committee on Cancer 5 and which include briefly:

Stage 0: LCIS or DCIS;

Stage I: invasive carcinoma ≤ 2 cm in diameter without nodal involvement;

Stage II: invasive carcinoma ≤ 5 cm in diameter with up to three involved axillary nodes, or invasive carcinoma > 5 cm without nodal involvement;

Stage III: invasive carcinoma ≤ 5 cm in diameter with four or more involved axillary nodes, or invasive carcinoma > 5 cm in diameter with nodal involvement, or invasive carcinoma with ≥ 10 involved axillary nodes, or invasive carcinoma with involvement of the ipsilateral internal mammary lymph nodes, or invasive carcinoma with skin involvement, chest wall fixation, or clinical inflammatory carcinoma;

Stage IV: any distant metastases.

The common standard international approach among pathologists for measuring tumour size was followed in this study. Accordingly, we used the largest diameter of the cut surface of the tumour, and we made much of the diameter of the invasive component. Therefore, we made use of the largest size of the invasive component on the cut surface.