Deciphering Invasive Breast Cancer Heterogeneity for Personalised Therapy

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Background and Aims: Characterisation of molecular and cellular heterogeneity in breast cancer (BC) is crucial to refine the prognostic classification of BC and understand the development of treatment resistance. This study investigated the hypotheses that: (i) molecular heterogeneity in terms of expression of BC stem cell (BCSC) markers, oestrogen receptor alpha (ER) and ER-related biomarkers, and the density of tumour-infiltrating lymphocytes (TILs) exists within individual tumours and also between different tumours, and (ii) assessment of such heterogeneity using high-throughput pathological techniques may help to predict tumour progression and patient outcomes. Materials and Methods: Primary invasive BC tissue specimens (n = 1948), including needle core biopsies (NCBs) and surgical excision specimens (SESs), lymph node (LN) metastases and recurrent tumour tissue samples were obtained for well-characterised cohorts of patients with primary invasive BC treated at Nottingham City Hospital between 1989 and 2018. Tissue microarrays were created and single and double immunohistochemistry, RNA in situ hybridisation (RNAscope), and haematoxylin and eosin staining were used to investigate inter-tumour and intra-tumour heterogeneity. The Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) cohort was used to assess mRNA and gene copy number changes. BC heterogeneity was quantified using a variety of pathologic assessments. Results: Analysis of cancer stem cell markers revealed BCSC markers exert different functions in BC and are co-expressed in varied combinations. High ALDH1A1 protein expression was associated with aggressive tumour features and poorer BC-specific survival (BCSS), especially in the luminal B and triple negative (TN) intrinsic subtypes. ALDH1A1 expression correlated positively with expression of other BCSC markers, including EpCAM and SOX9. High BMI-1 expression was associated with less aggressive tumour features and longer BCSS in ER+ tumours, but shorter BCSS in TNBC. BMI-1 expression correlated negatively with the BCSC markers ALDH1A1 and CD133 in ER+ tumour. Cells co-expressing ALDH1A1+, SOX9+ and EpCAM+ were observed in 12% of BC cases, in 1-10% of tumour cells. The ALDH1A1+SOX9+EpCAM+ BCSC phenotype was associated with advanced nodal stage, aggressive tumour features, ER- and HER2+ tumours, and poorer outcomes. Examination of ER expression in BC showed high concordance between ER and PgR expression at both the mRNA and protein levels. Approximately half of low ER (1-9%)-expressing NCBs were reclassified as ER-negative on the SESs (<1%); the NCBs of these cases were considered as ER false positives and repeat staining of low ER-expressing tumours is recommended. A high degree of intratumour ER heterogeneity was observed, which reflects the clinical significance of this receptor. Intra-tumoral heterogeneity in ER expression was observed between different regions of the same tumour, with varied patterns of ER staining intensity and distribution. A low ER heterogeneity score was associated with more aggressive tumour features and a shorter disease-free interval; a high ER heterogeneity score and the diffuse distribution pattern was associated with less aggressive features and a longer disease-free interval. The percentage of ER+ cells was higher in LN metastases compared to the paired primary tumours. High expression of the ER+ related marker. SLC39A6 was associated with good prognostic features, longer BCSS—especially in ER+ tumours—and expression of the ER-related proteins PgR, GATA3 and TFF1. Furthermore, high SLC39A6 expression was associated with longer BCSS and distant metastasis-free survival in patients who did not receive endocrine treatment, but not in patients who received endocrine treatment. The density of TILs did not vary across tumour blocks from the same case, but diff