Exploring the key molecular drivers of lymphovascular invasion in Invasive breast cancer
Publication Date
July 19, 2023
Creator
Abstract
BACKGROUND:
Lymphovascular invasion (LVI) plays a crucial role in breast cancer (BC) metastasis and presents as a reliable indicator of poor outcome in terms of distant metastasis and shorter survival. Therefore, accurate and reproducible assessment of LVI and an understanding of its molecular mechanisms can help guide management decision-making. However, molecular mechanisms of LVI are complex and overlap with other biological processes, such as metastasis and BC progression in general. Therefore, deciphering the molecular mechanisms of LVI and identifying its driver genes remain challenging. This study aimed to utilise several well-defined BC datasets, evaluating whether the LVI mechanisms in BC are controlled by distinct transcriptome and proteomic profiles, and to assess the roles of potential target genes using various molecular techniques.
METHODS:
In an attempt to discover new targets associated with LVI status, bioinformatics analysis was applied on multiple large cohorts of patients with BC, including the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) cohort (n = 1,980) and The Cancer Genome Atlas Breast Carcinoma (TCGA-BRCA; n = 854) as discovery datasets and the Breast Cancer Gene-Expression Miner (bc-GenExMiner) dataset (Affymetrix; n = 4,904) cohorts as a validation dataset. The differential mRNA expression was performed to identify key genes with respect to LVI status, which was previously defined based on morphology and immunohistochemistry (IHC) for endothelial markers. In addition, mass spectrometry of a well-defined subset of BC with well-characterised LVI status was utilised to identify target genes at the protein level. The targeted genes that were strongly associated with LVI at the transcriptomic levels (HSP90AA1, SLC9A3R1, NOP56 and HMGB3) were further investigated to confirm their prognostic value and association with LVI at the protein level utilising IHC and tissue microarrays of a large annotated early stage BC with long-term followup Nottingham cohort (n = 2,497). The protein expression of (HSP90AA1, SLC9A3R1 and NOP56) was correlated with clinicopathological parameters, a large panel of relevant biomarkers, and patient outcomes to identify clinically reliable LVI-associated genes. Further functional experiments followed this to explore the mechanistic insights of the most likely target gene (HMGB3) for LVI in BC to identify novel BC prognostic and predictive value towards personalised therapy in patients with early stage BC.
RESULTS:
Using the METABRIC and TCGA-BRCA datasets for the identification of molecular signatures strongly correlated with identified LVI, a set of 99 genes demonstrated significantly different expression patterns between LVI+ve and LVI–ve cases. Clustering analysis with this gene set further divided the cases into two molecular subtypes (subtypes 1 and 2), which were significantly associated with LVI status and patient outcome. The 10-year overall survival of subtype 2 was significantly worse than that of subtype 1. Expression of four individual genes strongly correlated with LVI. High expression of HSP90ΑA1 at both the mRNA and protein levels was significantly associated with characteristics of poor BC prognosis, including high grade, LVI positivity, poor Nottingham Prognostic Index (NPI) and positive expression of p53 and phosphoinositide-3-kinase, catalytic, alpha polypeptide (PIK3CA). In addition, outcome analysis revealed that high HSP90AA1 mRNA expression had significantly shorter BC-specific survival (BCSS) in the METABRIC and TCGA-BRCA datasets (hazard ratio (HR) = 1.38; 95% confidence interval [CI] = 1.16–1.65, p = 0.0004; and HR = 1.85; 95% CI = 1.318–2.61, p < 0.0001, respectively). The HSP90α protein expression was an independent predictor of shorter BCSS (HR = 1.38; 95% CI = 1.06–1.80, p = 0.018) and distant metastasis-free survival (HR = 1.32; 95% CI = 1.03–1.69, p = 0.027). High SLC9A3R1 mRNA and/or SLC9A3R1 protein expression was significantly correlated with aggressive tumour behaviour, LVI positivity, large tumour size, luminal B and human epidermal growth factor receptor 2 (HER2) positivity, high Ki67, and the NPI poor prognostic group. Both protein and mRNA expression of SLCA3R1 was significantly associated with PIK3CA and phosphorylated AKT. The SLC9A3R1 mRNA expression was higher in those tumours showing a copy number gain in both METABRIC and TCGA-BRCA (p < 0.0001). In the outcome analysis, SLC9A3R1 mRNA showed poor prognosis in bc-GenExMiner (HR = 1.59, 95% CI = 1.23–2.06, p = 0.0005), and high SLC9A3R1 protein expression was prognostic of shorter survival (HR = 1.52; 95% CI = 1.14–2.04, p = 0.005). High NOP56 mRNA and/or protein expression was significantly correlated with aggressive tumour behaviour, including high histological grade, larger tumour size, positive nodal status, LVI positivity, hormone receptor negativity, triple-negative BC positivity and NPI poor prognostic group. High NOP56 protein expression was associated with high Ki67 and significantly associated with PI3KCA and epidermal growth factor. A significant correlation between NOP56 protein and NOP56 mRNA was also observed (p < 0.001). High NOP56 mRNA correlated with shorter survival BCSS in the METABRIC cohort (p < 0.001, HR = 1.3, 95% CI = 1.1–1.5) and bc-GenExMiner (HR = 1.38, 95% CI = 1.16–1.65, p = 0.0003), and NOP56 protein presented a poor prognosis of BCSS (p = 0.003, HR = 1.4, 95% CI = 1.1–2.0). In both the METABRIC and TCGA-BRCA cohorts, high HMGB3 mRNA expression was significantly associated with the poor BC prognosis characteristics, including LVI positivity, high tumour grade, NPI poor prognostic group, high Ki67, hormone receptor negativity, HER2 positivity and phosphorylated AKT positivity. In addition, high HMGB3 mRNA expression was positively correlated with high level of PIK3CA. In the survival analysis, high HMGB3 mRNA was prognostic for shorter BCSS in LVI+ tumours in METABRIC (HR = 1.447; 95% CI = 1.023–2.05, p = 0.034) and TCGA-BRCA (HR = 2.826; 95% CI = 1.222–6.539, p = 0.015). Consistent with the transcriptomic results, HMGB3 knockdown reduced proliferation activity and led to a significant decrease in tumour cell adhesion, migration and transmigration ability.
CONCLUSIONS:
This investigation of independent large BC patient cohorts using several molecular techniques and bioinformatics analyses highlighted potential molecular and biological roles of LVI and their importance in BC progression, revealing the different aspects of BC metastasis and enhancing current knowledge. Moreover, the expression alteration of a group of genes and proteins may be responsible for the beginning or inhibition of LVI in invasive BC. This research showed that the clinicopathological index LVI in BC is linked to a unique transcriptome profile that may have prognostic significance. It identified a unique LVI profile with prognostic utility in BC. The proteins identified participate in various pathways manipulating the pre-LVI biological steps in BC, including adhesion, migration and invasion. In the future, the use of comprehensive and well-designed functional investigations with the help of several models and techniques may help further elucidate the roles played by each candidate target identified as controlling or inhibiting LVI status in patients with BC
Item Type
ethesis
Thesis Type
PhD
Supervisors
Subjects (LC)
Associated Schools / Departments
School of Medicine
eprints ID
73917
UoN Repository URI
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