Characterization Of Breast Cancer Intrinsic Subtypes In Relation To Risk Factors, Tumor Grade And Tumor Infiltrating Leukocytes In Western Kenya

ABSTRACT

Breast cancer contributes to 23% of all female cancers and is mostly developed among women aged 40-49 in Kenya. There is paucity of data explaining why breast cancer in Kenya and other African countries occurs at a younger age, although many risk factors were identified and studied in Western countries, there is a lack of research on the consistency of these risk factors among developing countries. Breast cancer seen in Africans is likely to be high-grade and hormone receptor negative, however profiling of breast cancer by hormonal receptor status is not documented because this is not routinely done in most Kenyan hospitals. Breast cancer in Kenya is uniquely aggressive and seem different for each individual. The explanation for this may be related to how an individual’s immune system mounts a response to cancer antigens. There was need to determine the type and density of immune cell infiltration in breast tumors of Kenyan women. This study was conducted at Moi Teaching and Referral Hospital (MTRH) which is located in Uasin Gishu County. The study determined the risk factors of breast cancer; characterize breast cancer into intrinsic subtypes, determining the type and density of TILs in tumor microenvironment and correlated this across grades and subtypes. A comparative cross-sectional study design was used to collect data from 160 participants who consented. Sixty nine breast cancer cases and 91 non cancer controls were consecutively enrolled from May 2011 to May 2013. Structured pre-tested, interviewer administered questionnaire was used to collect data on demographics, family history, age at first menarche, and number of pregnancies, breast feeding, use of contraceptives, smoking, alcohol consumption and other environmental factors. Tissue micro assays (TMAs) were constructed from all breast tissues then stained with heamatoxin and eosin for histological typing and grading. Immunohistochemical technique was used to stain for a panel of primary antibodies ER, PR, HER2, Ki67, CD4, CD8, CD20, CD68, CD163, and CD25. Immunohistochemistry (IHC) images were quantified with Aperio Image Analysis Tools software, output results were exported as an Excel file. Data was summarized using frequencies for categorical variables and median (IQR) for continuous/discrete variables. Multiple binary logistic regression was used to identify risk factors of cancer controlling for confounders. Ki67 and TILs markers were compared across grade and molecular subtype by Kruskal-Wallis followed by Dunn’s multiple comparison tests. Level of significance was set at p < 0.05. Marital status and environmental factors such as exposure to wood smoke are high level risk factors to breast. Alcohol consumption was a significant risk factors of breast cancer (p=0.029). The Kalenjin tribe were more likely to be cases compared to other tribes (OR; 95%CI: 3.192(0.661-15.404) though not statistically significant. Similarly, those using injection for contraceptive are more likely to be cases (OR; 95%CI: 4.499(0.735-27.545)). The mean age of the study population was 48.4 (SD 16.8). The tumors analyzed were heterogeneous by grade: grade I (5.8%), grade II (53.8%), and grade III (40.4%). Most patients presented with large tumors (>2.0cm) (80%). Invasive ductal carcinoma was the predominant (79%) histological type. Intrinsic subtypes were; luminal B (30.2%), basal/triple negative (TN) (34%), luminal A (26.4%) and HER2 (2%). There was a significant increase in percentage of tissue and alternative macrophages (CD68+, CD163+/M2 respectively) (p ≤0.0001) in cancer and non-cancer individuals. Cancer tissues showed an increase infiltration of CD4+ (helper) and CD25+ (inducible regulatory) T cells (p = 0.03; p=0.0001 respectively), CD8+ and CD20+ showed no significance. TNBC subtype had a much higher proliferative index (Ki67+) than the other intrinsic subtypes, there was no significant correlation between TIL type and density across subtype and tumor grade. Findings of the current study suggest sporadic genetic changes triggered by environmental, social and cultural changes are associated to the early onset of breast cancer. Routine staining for IHC4 clinical markers in breast cancer tissues will enable the identification of patient subgroups with different treatment requirements.