Chitra Joseph

The role of BUB and CDC proteins in low-grade breast cancers

BACKGROUND The drivers of neoplasia within low-grade luminal breast cancers remain undelineated. The BUB and CDC family are among kinase genes known recently to help to identify luminal breast cancers with poorer prognosis. Additionally, other CDC kinase genes (CDC42) are associated with luminal A breast cancers with good prognosis. We aimed to investigate the role of these kinases at the protein level within low-grade luminal breast cancers. METHODS The Nottingham Tenovus Primary Breast Cancer Series (n=1858) microarrays were immunostained for BUB (BUB1, BUB1B, BUB3) and CDC proteins (CDC2, CDC42) and expression correlated with clinicopathological and molecular variables and patient outcome (SPSS, version 22). FINDINGS On χ(2) analysis, cytoplasmic BUB1 and nuclear BUB3 were negatively associated with grade including pleomorphism, mitosis, and Nottingham Prognostic Index, whereas BUB1B was positively associated (p=0·05). BUB1 and BUB3 expression was positively correlated with oestrogen and progesterone receptor expression whereas BUB1B was negatively correlated (p=0·01). CDC42 had strong associations with tumour morphology within the low-grade luminal breast cancers, tubular and lobular (p=0·02). CDC42 nuclear expression revealed negative correlations with basal (CK5) and HER family biomarkers (p=0·02). By contrast, cytoplasmic CDC2 overexpression was associated with high-grade tumours (p=0·01). BUB1, BUB1B, and CDC42 showed significant associations (p=0·04) with breast-cancer-specific survival even at the 15-20-year range, indicating their long-term prognostic potential. INTERPRETATION These results suggest that BUB1, BUB3, and CDC42 are key kinases for low-grade luminal tumours whereas BUB1B and CDC2 kinases are preferentially expressed in high-grade disease. High protein expression of BUB1, BUB3, and CDC42 in low-grade breast cancers was associated with longer overall survival whereas lower expression resulted in poorer outcome. FUNDING Pathological Society of Great Britain and Northern Ireland, National Institute for Health Research.

Breast cancer intratumour heterogeneity: current status and clinical implications

Breast cancer (BC) is a heterogeneous disease that varies in presentation, morphological features, behaviour, and response to therapy. High‐throughput molecular profiling studies have revolutionised our understanding of BC heterogeneity, and have demonstrated that molecular profiles of tumours are variable not only between tumours, but also within individual tumours. Current evidence indicates that spatial and temporal intratumour heterogeneity of BC exists at levels beyond what are commonly expected. Intratumour heterogeneity poses critical challenges in the diagnosis, prediction of behaviour and management of BC. For instance, heterogeneous expression of oestrogen receptor, progesterone receptor and human epidermal growth factor receptor 2 can be seen not only in primary tumours between different regions, but also between primary tumours and their corresponding metastatic/recurrent lesions. The demonstration of molecularly distinct subclones within individual tumours may explain, at least in part, the mechanisms controlling the variable behaviour of BC, and may change our approach to BC sampling and treatment. In this review, BC intratumour heterogeneity is highlighted, with a special emphasis on the current knowledge pertaining to the relationship between intratumour heterogeneity and BC pathogenesis, evolution, and progression, with consideration of its impact on disease diagnosis, management, and the emergence of novel therapeutic targets. The key role of high‐throughput molecular and imaging techniques is also addressed.

Mediator complex (MED) 7: a biomarker associated with good prognosis in invasive breast cancer, especially ER+ luminal subtypes

BackgroundMediator complex (MED) proteins have a key role in transcriptional regulation, some interacting with the oestrogen receptor (ER). Interrogation of the METABRIC cohort suggested that MED7 may regulate lymphovascular invasion (LVI). Thus MED7 expression was assessed in large breast cancer (BC) cohorts to determine clinicopathological significance.MethodsMED7 gene expression was investigated in the METABRIC cohort (n = 1980) and externally validated using bc-GenExMiner v4.0. Immunohistochemical expression was assessed in the Nottingham primary BC series (n = 1280). Associations with clinicopathological variables and patient outcome were evaluated.ResultsHigh MED7 mRNA and protein expression was associated with good prognostic factors: low grade, smaller tumour size, good NPI, positive hormone receptor status (p < 0.001), and negative LVI (p = 0.04) status. Higher MED7 protein expression was associated with improved BC-specific survival within the whole cohort and ER+/luminal subgroup. Pooled MED7 gene expression data in the external validation cohort confirmed association with better survival, corroborating with the protein expression. On multivariate analysis, MED7 protein was independently predictive of longer BC-specific survival in the whole cohort and Luminal A subtype (p < 0.001).ConclusionsMED7 is an important prognostic marker in BC, particularly in ER+luminal subtypes, associated with improved survival and warrants future functional analysis.

Clinical and biological roles of Kelch-like family member 7 in breast cancer: a marker of poor prognosis

BackgroundThe functions of many proteins are tightly regulated with a complex array of cellular functions including ubiquitination. In cancer cells, aberrant ubiquitination may promote the activity of oncogenic pathways with subsequent tumour progression. Kelch-like family member 7 (KLHL7) is involved in the regulation of ubiquitination and may play a role in breast cancer (BC). Present study aims to evaluate the biological and clinical usefulness of KLHL7 in BC utilising large well-characterised cohorts with long-term follow-up.MethodsThe relationships between KLHL7 gene copy number alteration (CNA) and mRNA expression and clinicopathological variables and clinical outcomes were evaluated in 1980 patients from the METABRIC BC cohort. Prognostic significance of KLHL7 mRNA was validated using the Breast Cancer Gene-Expression Miner v4.0 datasets (n = 5206). KLHL7 protein expression was assessed using immunohistochemistry in a large annotated series of early-stage BC (n = 917) with long-term follow-up.ResultsKLHL7 CNA was significantly correlated with its mRNA expression. KLHL7 mRNA expression was higher in luminal B and basal-like molecular subtypes and in higher grade tumours. Increased KLHL7 protein expression was significantly correlated with features of aggressive phenotype including lymphovascular invasion, high histological grade, hormonal receptor negativity, high PIK3CA and p53 expression. Outcome analysis showed that high KLHL7 expression is an independent predictor of shorter survival (p = 0.0011).ConclusionsKLHL7 appears to play an important role in BC progression. High KLHL7 protein expression identified a subgroup of BC with aggressive behaviour and provided independent prognostic information.

Saccharomyces cerevisiae-like 1 (SEC14L1) is a prognostic factor in breast cancer associated with lymphovascular invasion

Lymphovascular invasion is strongly related to breast cancer metastasis. However, the underlying mechanisms of lymphovascular invasion and its driver molecules in breast cancer remain to be defined. In this study, we explore differential expression of genes in large molecularly characterized and clinically annotated datasets of invasive breast cancer patients (n = 8056) coupled with histological review and strict definition for lymphovascular invasion status. The METABRIC series was used to identify genes associated with lymphovascular invasion, as defined using hematoxylin and eosin staining supplemented by immunohistochemistry, at the genomic/transcriptomic levels. Saccharomyces cerevisiae-like 1 (SEC14L1) was identified as one of the most significant genes associated with lymphovascular invasion. The prognostic significance of SEC14L1 gene copy number and mRNA expression was further investigated in the METABRIC series and externally validated using the Breast Cancer Gene-Expression Miner v4.0. Protein expression of SEC14L1 was also assessed using immunohistochemistry in series of early stage breast cancer using tissue microarrays. SEC14L1 gene copy number gain was significantly associated with high histological grade and poor outcome. SEC14L1 mRNA expression showed positive association with higher grade, lymph node metastasis, and poor outcome. SEC14L1 protein overexpression was significantly associated with lymphovascular invasion (p < 0.0001), higher grade (p = 0.011), HER2 positivity (p = 0.036), and shorter survival (p = 0.00075). Our findings specify SEC14L1 as an independent prognostic factor in breast cancer. Its association, at both transcriptome and protein expression levels, with lymphovascular invasion and outcome could imply an important role in tumor progression. A further mechanistic insight into its molecular roles including potential therapeutic utility is warranted.

Heterogeneity of Tumour Infiltrating Lymphocytes (TILs) in breast cancer and its prognostic significance

Tumour‐infiltrating lymphocytes (TILs) in breast cancer (BC) provide prognostic and predictive information. The aim of this study was to assess the spatial and temporal heterogeneity of TILs in BC and its relationship with immune cell subtypes.

Clinicopathological and prognostic significance of Ras association and pleckstrin homology domains 1 (RAPH1) in breast cancer

BackgroundRas association and pleckstrin homology domains 1 (RAPH1) is involved in cytoskeleton regulation and re-epithelialisation in invasive carcinoma and, therefore, may play a key role in carcinogenesis and metastasis. We, herein, investigated the biological and clinical significance of RAPH1 in breast cancer using large annotated cohorts.MethodsThe clinicopathological and prognostic significance of RAPH1 was assessed at the genomic and transcriptomic levels using The Cancer Genome Atlas (TCGA) dataset (n = 1039) and the results were validated using the Molecular taxonomy of breast cancer international consortium (METABRIC) cohort (n = 1980). RAPH1 protein expression was evaluated by immunohistochemistry in a large, well-characterised cohort of early-stage breast cancer (n = 1040).ResultsIn both the TCGA and METABRIC cohorts, RAPH1 mRNA expression and RAPH1 copy number alteration were strongly correlated. RAPH1 mRNA overexpression was significantly correlated with high expression of adhesion and EMT markers including CDH1, TGFβ1 and CD44. RAPH1 mRNA overexpression was a significant predictor of a poor prognosis (Hazard ratio 3.88; p = 0.049). High RAPH1 protein expression was associated with higher grade tumours with high proliferation index, triple negative phenotype and high E-cadherin expression. High RAPH1 protein expression was an independent predictor of shorter survival (Hazard ratio 4.37; p = 0.037).ConclusionsHigh RAPH1 expression is correlated with aggressive breast cancer phenotypes and provides independent prognostic value in invasive breast cancer.

Exploring molecular pathways in low-grade oestrogen receptor positive breast cancer: a biomarker driven approach

Abstract Background The identification of the drivers of neoplasia and progression in low-grade oestrogen receptor (ER) positive luminal breast cancers compared with high-grade luminal breast cancers is essential. A surrogate biomarker profile is needed to distinguish between low-grade and high-grade cancers to help clinicians administer or avoid additional therapy. This study aimed to investigate molecular determinants of low-grade luminal breast cancers from a biomarker database. Methods 214 biomarkers from a well-annotated series of 1845 primary breast cancers were analysed for correlations with grade of breast cancer. Proteins with significant associations with grade 1 ER-positive cases (n=388) versus high-grade ER-positive cases (n=1183) were interrogated for pathway enrichment analysis (corrected for false discovery rate), with the STRING 10 platform (open access web-resource) incorporating Gene Ontology (GO) and KEGG. Findings Univariate analysis (p range=0·041–0·0001) identified 28 markers with significant association for grade 1 ER-positive tumours (eg, androgen receptor [AR], FHIT, FOXA1) and 54 with a significant negative association (eg, PTEN, GATA3, KAPNA1, p53). Pathways unifying the positively associated proteins in low-grade breast cancers revealed enrichments in intracellular steroid hormone receptor signalling (p=0·003), mammary gland development (p=0·008), AR binding (p=0·017) (GO). For negatively associated proteins, significant enrichments included cell proliferation (p=0·0002), radiation response (p=0·0003), T cell differentiation (p=0·004), and double-strand break repair (p=0·0004) (GO), and the HIF1α (p=0·00003), p53 (p=0·0016), ErbB (p=0·002), and JAK-STAT (p=0·01) (KEGG) pathways. Interpretation These preliminary findings are the first, to our knowledge, to unify biomarkers for grade-related analysis in breast cancer, with use of protein based data. Within the constraints of selection bias, data mining from immunohistochemistry of multiple biomarkers in relation to the features and behaviour of breast cancer hold promise. Funding Pathological Society of Great Britain and Ireland, National Institute for Health Research.