Introduction: There are two main morphological types of breast cancer, Invasive Ductal Carcinoma (IDC) and Invasive Lobular Carcinoma (ILC), which comprise up to 80% and 15% of all breast cancers, respectively. They arise from different evolutionary pathways and harbour distinct tumour phenotype resulting in differences in clinical behaviour. The tumour microenvironment including the breast stroma as one of the main constituents also plays a significant role in determining tumour behaviour. This thesis seeks to address various aspects of tumour development and progression in both IDC and ILC by taking into account the role of the neoplastic epithelium and the local microenvironment.
The progression of Ductal Carcinoma in situ (DCIS) to IDC marks a critical step in the evolution of breast cancer, wherein dynamic tumour-stroma interactions have been demonstrated to play an important role. The first aim of this study includes the use of gene expression profiling to study the transition from DCIS to IDC in the context of both the epithelial and the stromal breast compartments (Chapters 3-4).
In addition, the clinical and biological properties of ILC are also likely to be affected by the local microenvironment. This has been explored from the radiological, pathological and biological correlation to study tumour presentation, breast density and metastatic progression to the lymph nodes (Chapters 5-6).
Methods: Assessment of pathology features was a fundamental aspect of this study to ensure the correct classification and categorisation of all clinical samples used. Analysis of pathology reports to obtain clinical data relating to tumour diagnosis and standard molecular pathology techniques (tissue microarray (TMA) construction and immunohistochemistry (IHC)) were routinely employed. Gene expression profiling using the WG-DASL assay (Illumina) was performed on 87 FFPE samples derived from 17 patients consisting of matched IDC, DCIS and normal and cancer-associated stroma (Chapter 3). Candidate genes involved in the transition of DCIS to IDC were validated by quantitative Real Time-PCR (qRT-PCR), IHC and immunofluorescence (IF) and a further investigation of two genes (SOX10 and SFRP1) was performed in triple-negative (TN) tumours (Chapter 4). A series of 147 ILC cases with detailed radiological imaging data was analysed by pathology review, IHC on TMAs and statistical analysis to correlate with the radiological data (Chapter 5). A series of 88 ILC was then studied using similar methods to investigate progression of disease to lymph nodes (Chapter 6).
Results: WG-DASL analysis demonstrated that the neoplastic epithelium underwent more gene expression changes than the stroma during progression from DCIS to IDC. Genes of interest such as SFRP1 and extracellular matrix-related genes (e.g. COL11A1, COL5A2 and MMP13) were down-and up-regulated during transition, respectively. Interestingly, COL11A1 was shown to be produced by the tumour epithelial cells to significantly higher levels than DCIS and the immediate adjacent stroma suggesting that this, among other ECM proteins, might play a role in local invasion of breast cancer cells. In addition, it was confirmed by qRT-PCR and meta-analysis on publically available microarray data, that SFRP1 and SOX10 were up-regulated in a proportion of tumours and breast cancer cell lines of triple-negative/basal-like phenotype.
Several clinical-pathological features of ILC were significantly associated with either the mammographic presentation of ILC or with an increased breast density in the non-neoplastic breast parenchyma of these patients. Of particular interest were, for instance, the patient's age at diagnosis, tumour size, the presence and extent of ILC precursor lesions and columnar cell change, the extent of lymphocytic infiltrate and the presence of a predominantly collagenous tumour stroma. A number of histopathological characteristics correlated with an increased frequency of lymph node metastasis including moderate levels of tumour infiltrating lymphocytes and HER2 over-expression. Although proliferation, as assessed by KI-67 staining, was not differentially expressed between primary ILC with or without LN metastases, it was significantly increased in lymph node metastases when compared to matched primary tumours.
Conclusions: Differential expression of several genes related to extracellular matrix remodelling such as COL11A1, COL5A2 and MMP13 could be involved in facilitating the transition from DCIS to IDC and may thus mark a critical point in disease development. In addition, loss of expression of SFRP1 may play a role in progression of luminal and HER2 tumours but conversely, and along with SOX10, play an important role in the biology of the triple negative/basal-like phenotype. Regarding ILC, this study contributed to understanding the correlation between the mammographic presentation of ILC and the clinical and pathological features of the tumour and its microenvironment that might contribute to the tumours being missed by mammography and hence a delayed diagnosis. A consequence of which might be tumour progression, and through the analysis of ILC with or without metastasis to lymph nodes an association was found between the extent of tumour infiltrating lymphocytes and an increased frequency of lymph node metastasis. This gives weight to the role that the tumour microenvironment plays in the metastatic progression of this special type of breast cancer.