A Case Series Exploration of Multi-Regional Expression Heterogeneity in Triple-Negative Breast Cancer Patients.

Extensive intratumoral heterogeneity (ITH) is believed to contribute to therapeutic failure and tumor recurrence, as treatment-resistant cell clones can survive and expand. However, little is known about ITH in triple-negative breast cancer (TNBC) because of the limited number of single-cell sequencing studies on TNBC. In this study, we explored ITH in TNBC by evaluating gene expression-derived and imaging-derived multi-region differences within the same tumor. We obtained tissue specimens from 10 TNBC patients and conducted RNA sequencing analysis of 2-4 regions per tumor. We developed a novel analysis framework to dissect and characterize different types of variability: between-patients (inter-tumoral heterogeneity), between-patients across regions (inter-tumoral and region heterogeneity), and within-patient, between-regions (regional intratumoral heterogeneity). We performed a Bayesian changepoint analysis to assess and classify regional variability as low (convergent) versus high (divergent) within each patient feature (TNBC and PAM50 subtypes, immune, stroma, tumor counts and tumor infiltrating lymphocytes). Gene expression signatures were categorized into three types of variability: between-patients (108 genes), between-patients across regions (183 genes), and within-patients, between-regions (778 genes). Based on the between-patient gene signature, we identified two distinct patient clusters that differed in menopausal status. Significant intratumoral divergence was observed for PAM50 classification, tumor cell counts, and tumor-infiltrating T cell abundance. Other features examined showed a representation of both divergent and convergent results. Lymph node stage was significantly associated with divergent tumors. Our results show extensive intertumoral heterogeneity and regional ITH in gene expression and image-derived features in TNBC. Our findings also raise concerns regarding gene expression based TNBC subtyping. Future studies are warranted to elucidate the role of regional heterogeneity in TNBC as a driver of treatment resistance.

Ran GTPase is an independent prognostic marker in malignant melanoma which promotes tumour cell migration and invasion.

AIMS: Ran GTPase is involved in nucleocytoplasmic shuttling of proteins and is overexpressed in several cancers. The expression of Ran in malignant melanoma (MM) and its functional activity have not been described and were investigated in this study. METHODS: The prognostic value of Ran expression was tested in a series of 185 primary cutaneous MM cases using immunohistochemistry. The functional activity of Ran was investigated in the two melanoma cell lines. Ran expression was knocked down using two siRNAs and the effect on the expression of the c-Met oncogene, a potential downstream target of Ran, was tested. Functional effects of Ran knockdown on cell motility and cell proliferation were also assessed. RESULTS: Positive Ran expression was seen in 12.4% of MM and was associated with advanced clinical stage and greater Breslow thickness. Positive expression was an independent marker of shorter overall survival (p=0.023). Knockdown of Ran results in decreased expression of c-Met and the downstream c-met signalling targets ERK1/2. There was a significant reduction in cell migration (p<0.001) and cell invasion (p<0.001). c-Met knockdown decreased the expression of Ran through MAPK and PI3K-AKT in A375 cell line, inhibited the cell viability and migration of both A375 and G361 melanoma cell lines while invasion was enhanced. CONCLUSION: Ran is a poor prognostic marker in cutaneous MM. It upregulates expression of the oncogene c-Met and, possibly through this, it promotes cell motility which may in turn promote metastasis.

Prognostic significance of heat shock protein 90AA1 (HSP90α) in invasive breast cancer.

AIMS: The mechanisms that drive breast cancer (BC) progression and poor outcome are not fully understood. The human heat shock protein 90 alpha family class A member 1 (HSP90α) encoded by the HSP90ΑA1 gene has a vital role in cellular responses to stress and is implicated in the development and progression of many cancers. The current study aims to explore the clinical and prognostic importance of HSP90α in BC. METHODS: The Molecular Taxonomy of Breast Cancer International Consortium (n=1980); The Cancer Genome Atlas (n=1097) and the Breast Cancer Gene-Expression Miner (Bc-GenExMiner) BC datasets (n=5056) were used to evaluate HSP90ΑA1 mRNA expression. HSP90α protein expression was further assessed using immunohistochemistry in a large (n=911) well-characterised BC series. The association between mRNA and protein expressions with other clinicopathological parameters and outcome was analysed. RESULTS: High expression of HSP90ΑA1 both at the mRNA and protein levels was significantly associated with characteristics of BC poor prognosis, including high grade, lymphovascular invasion, poor Nottingham Prognostic Index and positive expression of p53 and PIK3CA. Outcome analysis revealed that high HSP90α protein expression is an independent predictor of shorter BC-specific survival. CONCLUSION: HSP90α can be used as a potential prognostic marker in BC. Further mechanistic studies are warranted to determine the underlying molecular mechanisms mediated by HSP90α in BC.

Hypoxia Drives Centrosome Amplification in Cancer Cells via HIF1α-dependent Induction of Polo-Like Kinase 4.

Centrosome amplification (CA) has been implicated in the progression of various cancer types. Although studies have shown that overexpression of PLK4 promotes CA, the effect of tumor microenvironment on polo-like kinase 4 (PLK4) regulation is understudied. The aim of this study was to examine the role of hypoxia in promoting CA via PLK4. We found that hypoxia induced CA via hypoxia-inducible factor-1α (HIF1α). We quantified the prevalence of CA in tumor cell lines and tissue sections from breast cancer, pancreatic ductal adenocarcinoma (PDAC), colorectal cancer, and prostate cancer and found that CA was prevalent in cells with increased HIF1α levels under normoxic conditions. HIF1α levels were correlated with the extent of CA and PLK4 expression in clinical samples. We analyzed the correlation between PLK4 and HIF1A mRNA levels in The Cancer Genome Atlas (TCGA) datasets to evaluate the role of PLK4 and HIF1α in breast cancer and PDAC prognosis. High HIF1A and PLK4 levels in patients with breast cancer and PDAC were associated with poor overall survival. We confirmed PLK4 as a transcriptional target of HIF1α and demonstrated that in PLK4 knockdown cells, hypoxia-mimicking agents did not affect CA and expression of CA-associated proteins, underscoring the necessity of PLK4 in HIF1α-related CA. To further dissect the HIF1α-PLK4 interplay, we used HIF1α-deficient cells overexpressing PLK4 and showed a significant increase in CA compared with HIF1α-deficient cells harboring wild-type PLK4. These findings suggest that HIF1α induces CA by directly upregulating PLK4 and could help us risk-stratify patients and design new therapies for CA-rich cancers. IMPLICATIONS: Hypoxia drives CA in cancer cells by regulating expression of PLK4, uncovering a novel HIF1α/PLK4 axis.

Oestrogen-regulated protein SLC39A6: a biomarker of good prognosis in luminal breast cancer.

PURPOSE: The outcome of the luminal oestrogen receptor-positive (ER +) subtype of breast cancer (BC) is highly variable and patient stratification needs to be refined. We assessed the prognostic significance of oestrogen-regulated solute carrier family 39 member 6 (SLC39A6) in BC, with emphasis on ER + tumours. MATERIALS AND METHODS: SLC39A6 mRNA expression and copy number alterations were assessed using the METABRIC cohort (n = 1980). SLC39A6 protein expression was evaluated in a large (n = 670) and annotated series of early-stage (I-III) operable BC using tissue microarrays and immunohistochemistry. The associations between SLC39A6 expression and clinicopathological parameters, patient outcomes and other ER-related markers were evaluated using Chi-square tests and Kaplan-Meier curves. RESULTS: High SLC39A6 mRNA and protein expression was associated with features characteristic of less aggressive tumours in the entire BC cohort and ER + subgroup. SLC39A6 protein expression was detected in the cytoplasm and nuclei of the tumour cells. High SLC39A6 nuclear expression and mRNA levels were positively associated with ER + tumours and expression of ER-related markers, including the progesterone receptor, forkhead box protein A1 and GATA binding protein 3. In the ER + luminal BC, high SLC39A6 expression was independently associated with longer BC-specific survival (BCSS) (P = 0.015, HR 0.678, 95% CI 0.472‒0.972) even in those who did not receive endocrine therapy (P = 0.001, HR 0.701, 95% CI 0.463‒1.062). CONCLUSION: SLC39A6 may be prognostic for a better outcome in ER + luminal BC. Further functional studies to investigate the role of SLC39A6 in ER + luminal BC are warranted.

The nucleolar-related protein Dyskerin pseudouridine synthase 1 (DKC1) predicts poor prognosis in breast cancer.

BACKGROUND: Hypertrophy of the nucleolus is a distinctive cytological feature of malignant cells and corresponds to aggressive behaviour. This study aimed to identify the key gene associated with nucleolar prominence (NP) in breast cancer (BC) and determine its prognostic significance. METHODS: From The Cancer Genome Atlas (TCGA) cohort, digital whole slide images identified cancers having NP served as label and an information theory algorithm was applied to find which mRNA gene best explained NP. Dyskerin Pseudouridine Synthase 1 (DKC1) was identified. DKC1 expression was assessed using mRNA data of Molecular Taxonomy of Breast Cancer International Consortium (METABRIC, n = 1980) and TCGA (n = 855). DKC1 protein expression was assessed using immunohistochemistry in Nottingham BC cohort (n = 943). RESULTS: Nuclear and nucleolar expressions of DKC1 protein were significantly associated with higher tumour grade (p < 0.0001), high nucleolar score (p < 0.001) and poor Nottingham Prognostic Index (p < 0.0001). High DKC1 expression was associated with shorter BC-specific survival (BCSS). In multivariate analysis, DKC1 mRNA and protein expressions were independent risk factors for BCSS (p < 0.01). CONCLUSION: DKC1 expression is strongly correlated with NP and its overexpression in BC is associated with unfavourable clinicopathological characteristics and poor outcome. This has been a detailed example in the correlation of phenotype with genotype.

Molecular Complexity of Lymphovascular Invasion: The Role of Cell Migration in Breast Cancer as a Prototype.

Lymphovascular invasion (LVI) is associated with poor outcome in breast cancer (BC); however, its underlying mechanisms remain ill-defined. LVI in BC develops through complex molecular pathways involving not only the interplay with the surrounding microenvironment along with endothelial cells lining the lymphovascular spaces but also changes in the malignant epithelial cells with the acquisition of more invasive and migration abilities. In this review, we focus on the key features that enable tumour cell detachment from the primary niche, their migration and interaction with the surrounding microenvironment as well as the crosstalk with the vascular endothelial cells, which eventually lead to intravasation of tumour cells and LVI. Intravascular tumour cell survival and migration, their distant site extravasation, stromal invasion and growth are part of the metastatic cascade. Cancer cell migration commences with loss of tumour cells' cohesion initiating the invasion and migration processes which are usually accompanied by the accumulation of specific cellular and molecular changes that enable tumour cells to overcome the blockades of the extracellular matrix, spread into surrounding tissues and interact with stromal cells and immune cells. Thereafter, tumour cells migrate further via interacting with lymphovascular endothelial cells to penetrate the vessel wall leading ultimately to intravasation of cancer cells. Exploring the potential factors influencing cell migration in LVI can help in understanding the underlying mechanisms of LVI to identify targeted therapy in BC.

The prognostic significance of BMI1 expression in invasive breast cancer is dependent on its molecular subtypes.

PURPOSE: BMI1, which is a major component of the polycomb group complex 1, is an essential epigenetic repressor of multiple regulatory genes and has been identified as a cancer stem cell (CSC) marker in several cancers. However, its role in breast cancer (BC) remains to be defined. In this study, we have evaluated the prognostic significance of BMI1 among the different molecular subtypes and assessed its association with other breast CSC markers (BCSC). MATERIAL AND METHOD: BMI1 copy number and mRNA was assessed in large and well-characterised cohorts of early-stage BC patients [METABRIC (n = 1980) and the Bc-GenExMiner (n = 9616) databases]. BMI1 protein expression was assessed using tissue microarray and immunohistochemistry in a cohort of 870 invasive BC patients with long-term outcome data and the expression of a panel of BCSC markers was monitored. RESULT: BMI1 expression, prognostic significance and its association with BCSC markers were differed between molecular classes. In the luminal oestrogen receptor-positive (ER+) BC, BMI1 showed significantly higher expression compared to ER- tumours. BMI1 showed positive correlation with favourable prognostic features and it was negatively associated with the expression of key BCSC markers (ALDH1A1, CD24, CD44, CD133, SOX10 and SOX9). High expression of BMI1 was associated with longer breast cancer-specific survival (BCSS) independent of other prognostic variables. In the basal triple negative BC subtype, BMI1 expression showed positive association with CD133 and SOX10 and it was significantly associated with shorter BCSS. CONCLUSION: High BMI1 expression is associated with clinicopathological variables and outcome in BC. However, this association is dependent on the molecular subtypes. Further functional assessment to detect its underlying mechanistic roles in BC subtypes is warranted.

The prognostic significance of ALDH1A1 expression in early invasive breast cancer.

AIMS: Aldehyde dehydrogenase family 1 member A1 (ALDH1A1) is reportedly a key ALDH isozyme linked to the cancer stem cells (CSC) of many solid tumours, where it is involved in self-renewal, differentiation and self-protection. In this study, the prognostic significance of ALDH1A1 expression in early invasive breast cancer (BC) and its role as a BC stem cell (BCSC) were evaluated. METHODS AND RESULTS: ALDH1A1 expression was assessed, using immunohistochemistry and tissue microarrays, in a large well-characterised BC cohort. ALDH1A1 mRNA expression was also assessed at transcriptomic levels, utilising data from the Molecular Taxonomy of Breast Cancer International Consortium. The associations of ALDH1A1 with clinicopathological parameters, other stem cell markers and patient outcomes were determined. ALDH1A1 was expressed in 71% of BC cases at both the protein and mRNA levels. High ALDH1A1 expression was associated with poor prognostic features, including high grade, poor Nottingham Prognostic Index (NPI), lymph node metastasis and highly proliferative ER+ (luminal B) and triple-negative (TNBC) subtypes. ALDH1A1 expression was positively correlated with the expression of CD44, CD24, TWIST, SOX9, EPCAM and CD133. The high immunoexpression of ALDH1A1 was significantly associated with poor BC-specific survival (P < 0.001), and specifically in the luminal B and TNBC subtypes (P = 0.042 and P = 0.003, respectively). The immunoexpression of ALDH1A1 was an independent predictor of poor prognosis (P = 0.015). CONCLUSIONS: ALDH1A1, as assessed using immunohistochemistry, seems to act as a BCSC marker associated not only with other BCSC markers but also with poor prognostic characteristics and poor outcomes, particularly in the luminal B and TNBC subtypes.

A novel prognostic two-gene signature for triple negative breast cancer.

The absence of a robust risk stratification tool for triple negative breast cancer (TNBC) underlies imprecise and nonselective treatment of these patients with cytotoxic chemotherapy. This study aimed to interrogate transcriptomes of TNBC resected samples using next generation sequencing to identify novel biomarkers associated with disease outcomes. A subset of cases (n = 112) from a large, well-characterized cohort of primary TNBC (n = 333) were subjected to RNA-sequencing. Reads were aligned to the human reference genome (GRCH38.83) using the STAR aligner and gene expression quantified using HTSEQ. We identified genes associated with distant metastasis-free survival and breast cancer-specific survival by applying supervised artificial neural network analysis with gene selection to the RNA-sequencing data. The prognostic ability of these genes was validated using the Breast Cancer Gene-Expression Miner v4. 0 and Genotype 2 outcome datasets. Multivariate Cox regression analysis identified a prognostic gene signature that was independently associated with poor prognosis. Finally, we corroborated our results from the two-gene prognostic signature by their protein expression using immunohistochemistry. Artificial neural network identified two gene panels that strongly predicted distant metastasis-free survival and breast cancer-specific survival. Univariate Cox regression analysis of 21 genes common to both panels revealed that the expression level of eight genes was independently associated with poor prognosis (p < 0.05). Adjusting for clinicopathological factors including patient's age, grade, nodal stage, tumor size, and lymphovascular invasion using multivariate Cox regression analysis yielded a two-gene prognostic signature (ACSM4 and SPDYC), which was associated with poor prognosis (p < 0.05) independent of other prognostic variables. We validated the protein expression of these two genes, and it was significantly associated with patient outcome in both independent and combined manner (p < 0.05). Our study identifies a prognostic gene signature that can predict prognosis in TNBC patients and could potentially be used to guide the clinical management of TNBC patients.

Combined HER3-EGFR score in triple-negative breast cancer provides prognostic and predictive significance superior to individual biomarkers.

Epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 3 (HER3) have been investigated as triple-negative breast cancer (TNBC) biomarkers. Reduced EGFR levels can be compensated by increases in HER3; thus, assaying EGFR and HER3 together may improve prognostic value. In a multi-institutional cohort of 510 TNBC patients, we analyzed the impact of HER3, EGFR, or combined HER3-EGFR protein expression in pre-treatment samples on breast cancer-specific and distant metastasis-free survival (BCSS and DMFS, respectively). A subset of 60 TNBC samples were RNA-sequenced using massive parallel sequencing. The combined HER3-EGFR score outperformed individual HER3 and EGFR scores, with high HER3-EGFR score independently predicting worse BCSS (Hazard Ratio [HR] = 2.30, p = 0.006) and DMFS (HR = 1.78, p = 0.041, respectively). TNBCs with high HER3-EGFR scores exhibited significantly suppressed ATM signaling and differential expression of a network predicted to be controlled by low TXN activity, resulting in activation of EGFR, PARP1, and caspases and inhibition of p53 and NFκB. Nuclear PARP1 protein levels were higher in HER3-EGFR-high TNBCs based on immunohistochemistry (p = 0.036). Assessing HER3 and EGFR protein expression in combination may identify which adjuvant chemotherapy-treated TNBC patients have a higher risk of treatment resistance and may benefit from a dual HER3-EGFR inhibitor and a PARP1 inhibitor.

The prognostic significance of wild-type isocitrate dehydrogenase 2 (IDH2) in breast cancer.

BACKGROUND: Lymphovascular invasion (LVI) is a prerequisite step in breast cancer (BC) metastasis. We have previously identified wild-type isocitrate dehydrogenase 2 (IDH2) as a key putative driver of LVI. Thus, we explored the prognostic significance of IDH2 at transcriptome and protein expression levels in pre-invasive and invasive disease. METHODS: Utlising tissue microarrays from a large well annotated BC cohort including ductal carcinoma in situ and invasive breast cancer (IBC), IDH2 was assessed at the transcriptomic and proteomic level. The associations between clinicopathological factors including LVI status, prognosis and the expression of IDH2 were evaluated. RESULTS: In pure DCIS and IBC, high IDH2 protein expression was associated with features of aggressiveness including high nuclear grade, larger size, comedo necrosis and hormonal receptor negativity and LVI, higher grade, larger tumour size, high NPI, HER2 positivity, and hormonal receptor negativity, respectively. High expression of IDH2 either in mRNA or in protein levels was associated with poor patient's outcome in both DCIS and IBC. Multivariate analysis revealed that IDH2 protein expression was an independent risk factor for shorter BC specific-survival. CONCLUSION: Further functional studies to decipher the role of IDH2 and its mechanism of action as a driver of BC progression and LVI are warranted.

Prognostic significance of nucleolar assessment in invasive breast cancer.

AIMS: Nucleolar morphometric features have a potential role in the assessment of the aggressiveness of many cancers. However, the role of nucleoli in invasive breast cancer (BC) is still unclear. The aims of this study were to investigate the optimal method for scoring nucleoli in IBC and their prognostic significance, and to refine the grading of breast cancer (BC) by incorporating nucleolar score. METHODS AND RESULTS: Digital images acquired from haematoxylin and eosin-stained sections from a large BC cohort were divided into training (n = 400) and validation (n = 1200) sets for use in this study. Four different assessment methods were evaluated in the training set to identify the optimal method associated with the best performance and significant prognostic value. These were: (i) a modified Helpap method; (ii) counting prominent nucleoli (size ≥2.5 µm) in 10 field views (FVs); (iii) counting prominent nucleoli in five FVs; and (iv) counting prominent nucleoli in one FV. The optimal method was applied to the validation set and to an external validation set, i.e. data from The Cancer Genome Atlas (n = 743). Scoring prominent nucleoli in five FVs showed the highest interobserver concordance rate (intraclass correlation coefficient of 0.8) and a significant association with BC-specific survival (P < 0.0001). A high nucleolar score was associated with younger age, larger tumour size, and higher grade. Incorporation of nucleolar score in the Nottingham grading system resulted in a higher significant association with survival than the conventional grade. CONCLUSIONS: Quantification of nucleolar prominence in five FVs is a cost-efficient and reproducible morphological feature that can predict BC behaviour and can provide an alternative to pleomorphism to improve BC grading performance.

Prognostic significance of KN motif and ankyrin repeat domains 1 (KANK1) in invasive breast cancer.

BACKGROUND: KN motif and ankyrin repeat domains 1 (KANK1) plays an important role in cytoskeleton maintenance and contributes to the regulation of cell proliferation, adhesion and apoptosis. KANK1 is involved in progression of a variety of solid tumours; however, its role in invasive breast cancer (BC) remains unknown. This study aims to evaluate the clinicopathological and prognostic value of KANK1 expression in operable BC. METHODS: KANK1 expression was assessed at the transcriptomic level using multiple BC cohorts; the Molecular Taxonomy of BC International Consortium cohort (METABRIC; n = 1980), The Cancer Genome Atlas BC cohort (TCGA; n = 949) and the publicly available BC transcriptomic data hosted by BC Gene-Expression Miner (bc-GenExMiner v4.0) and Kaplan-Meier plotter?. The Nottingham BC cohort (n = 1500) prepared as tissue microarrays was used to assess KANK1 protein expression using immunohistochemistry (IHC). The association between clinicopathological variables and patient outcome was investigated. RESULTS: In the METABRIC cohort, high expression of KANK1 mRNA was associated with characteristics of good prognosis including lower grade, absence of lymphovascular invasion and HER2 negativity (all; p < 0.001) and with better outcome [p = 0.006, Hazards ratio, (HR) 0.70, 95% CI 0.54-0.91]. High KANK1 protein expression was correlated with smaller tumour size and HER2 negativity, and better outcome in terms of longer breast cancer-specific survival [p = 0.013, HR 0.7, 95% CI 0.536-0.893] and time to distant metastasis [p = 0.033, HR 0.65, 95% CI 0.51-0.819]. CONCLUSION: These results supported that upregulation of KANK1 works as a tumour suppressor gene in BC and is associated with improved patients' outcomes.

Clinicopathological significance of lipocalin 2 nuclear expression in invasive breast cancer.

PURPOSE: The epithelial-mesenchymal transition (EMT) plays a key role in breast cancer progression and metastasis. Lipocalin 2 (LCN2) is involved in the regulation of EMT. The aim of this study was to investigate the clinicopathological significance of LCN2 expression in breast cancer. METHODS: The expression of LCN2 protein was immunohistochemically assessed in two well-characterised annotated cohorts of breast cancer (discovery cohort, n = 612; validation cohort, n = 1363). The relationship of LCN2 expression and subcellular location with the clinicopathological factors and outcomes of patients was analysed. RESULTS: Absent or reduced nuclear LCN2 expression was associated with features of aggressive behaviour, including high histological grade, high Nottingham Prognostic Index, high Ki67 labelling index, hormone receptor negativity and human epidermal growth factor receptor 2 positivity. The high cytoplasmic expression of LCN2 was correlated with lymph node positivity. The nuclear downregulation of LCN2 was correlated with the overexpression of EMT associated proteins (N-cadherin and Twist-related protein 2) and basal biomarkers (cytokeratin 5/6 and epidermal growth factor receptor). Unlike the cytoplasmic expression of LCN2, the loss of nuclear expression was a significant predictor of poor outcome. The combinatorial expression tumours with high cytoplasmic and low nuclear expression were associated with the worst prognosis. CONCLUSIONS: Tumour cell expression of LCN2 plays a role in breast cancer progression with loss of its nuclear expression which is associated with aggressive features and poor outcome. Further functional analysis is warranted to confirm the relationship between the subcellular localisation LCN2 and behaviour of breast cancer.

Retinoid X receptor gamma (RXRG) is an independent prognostic biomarker in ER-positive invasive breast cancer.

BACKGROUND: Retinoid X Receptor Gamma (RXRG) is a member of the nuclear receptor superfamily and plays a role in tumour suppression. This study aims to explore the prognostic significance of RXRG in breast cancer. METHODS: Primary breast cancer tissue microarrays (n = 923) were immuno-stained for RXRG protein and correlated with clinicopathological features, and patient outcome. RESULTS: Nuclear RXRG expression was significantly associated with smaller tumour size (p = 0.036), lower grade (p < 0.001), lobular histology (p = 0.016), lower Nottingham Prognostic Index (p = 0.04) and longer breast cancer-specific survival (p < 0.001), and longer time to distant metastasis (p = 0.002). RXRG expression showed positive association with oestrogen receptor (ER)-related biomarkers: GATA3, FOXA1, STAT3 and MED7 (all p < 0.001) and a negative correlation with the Ki67 proliferation marker. Multivariate analysis demonstrated RXRG protein as an independent predictor of longer breast cancer-specific survival and distant metastasis-free survival. In the external validation cohorts, RXRG expression was associated with improved patients' outcome (p = 0.025). In ER-positive tumours, high expression of RXRG was associated with better patient outcome regardless of adjuvant systemic therapy. ER signalling pathway was the top predicted master regulator of RXRG protein expression (p = 0.005). CONCLUSION: This study provides evidence for the prognostic value of RXRG in breast cancer particularly the ER-positive tumours.

Machine learning-based prediction of breast cancer growth rate in vivo.

BACKGROUND: Determining the rate of breast cancer (BC) growth in vivo, which can predict prognosis, has remained elusive despite its relevance for treatment, screening recommendations and medicolegal practice. We developed a model that predicts the rate of in vivo tumour growth using a unique study cohort of BC patients who had two serial mammograms wherein the tumour, visible in the diagnostic mammogram, was missed in the first screen. METHODS: A serial mammography-derived in vivo growth rate (SM-INVIGOR) index was developed using tumour volumes from two serial mammograms and time interval between measurements. We then developed a machine learning-based surrogate model called Surr-INVIGOR using routinely assessed biomarkers to predict in vivo rate of tumour growth and extend the utility of this approach to a larger patient population. Surr-INVIGOR was validated using an independent cohort. RESULTS: SM-INVIGOR stratified discovery cohort patients into fast-growing versus slow-growing tumour subgroups, wherein patients with fast-growing tumours experienced poorer BC-specific survival. Our clinically relevant Surr-INVIGOR stratified tumours in the discovery cohort and was concordant with SM-INVIGOR. In the validation cohort, Surr-INVIGOR uncovered significant survival differences between patients with fast-growing and slow-growing tumours. CONCLUSION: Our Surr-INVIGOR model predicts in vivo BC growth rate during the pre-diagnostic stage and offers several useful applications.

Prognostic Role of Androgen Receptor in Triple Negative Breast Cancer: A Multi-Institutional Study.

BACKGROUND: The androgen receptor (AR) has emerged as a potential therapeutic target for AR-positive triple-negative breast cancer (TNBC). However, conflicting reports regarding AR's prognostic role in TNBC are putting its usefulness in question. Some studies conclude that AR positivity indicates a good prognosis in TNBC, whereas others suggest the opposite, and some show that AR status has no significant bearing on the patients' prognosis. METHODS: We evaluated the prognostic value of AR in resected primary tumors from TNBC patients from six international cohorts {US (n = 420), UK (n = 239), Norway (n = 104), Ireland (n = 222), Nigeria (n = 180), and India (n = 242); total n = 1407}. All TNBC samples were stained with the same anti-AR antibody using the same immunohistochemistry protocol, and samples with ≥1% of AR-positive nuclei were deemed AR-positive TNBCs. RESULTS: AR status shows population-specific patterns of association with patients' overall survival after controlling for age, grade, population, and chemotherapy. We found AR-positive status to be a marker of good prognosis in US and Nigerian cohorts, a marker of poor prognosis in Norway, Ireland and Indian cohorts, and neutral in UK cohort. CONCLUSION: AR status, on its own, is not a reliable prognostic marker. More research to investigate molecular subtype composition among the different cohorts is warranted.

Collagen (XI) alpha-1 chain is an independent prognostic factor in breast ductal carcinoma in situ.

Collagen11A1 (COL11A1) is a fibrillary type collagen constituting a minor component of the extracellular matrix and plays role in tissue tensile strength. Overexpression of COL11A1 expression is associated with aggressive behavior and poor outcome in several human malignancies. In this study, we evaluated the association between COL11A1 expression and clinicopathological parameters of the breast ductal carcinoma in situ (DCIS) and its prognostic value. COL11A1 protein expression was assessed immunohistochemically in a large well-characterized cohort of DCIS including pure (n = 776) and DCIS associated with invasive carcinoma (DCIS-mixed, n = 239). COL11A1 expression was assessed in tumor cells and surrounding stromal cells, and correlated with clinicopathological parameters, immunoprofile and disease outcome. In pure DCIS, high COL11A1 expression was observed in tumor cells and surrounding stromal cells in 25 and 13% of cases, respectively. Higher COL11A1 expression within the stromal cells was associated with hormone receptor negative, HER2 enriched and triple negative molecular subtypes and showed a positive linear correlation with proliferation index, dense tumor infiltrating lymphocytes and hypoxia-inducible factor 1 alpha. COL11A1 expression in tumor and stromal cells was significantly higher in DCIS associated with invasive carcinoma than in pure DCIS, and within the DCIS-mixed cohort, the invasive component showed higher COL11A1 expression than the DCIS component (all, p < 0.0001). Overexpression of stromal COL11A1 was an independent predictor of shorter local recurrence-free interval for all recurrences (HR = 13.2, 95% CI = 6.9-25.4, p < 0.0001) and for invasive recurrences (HR = 11.2, 95% CI = 4.9-25.8, p < 0.0001). When incorporated with other risk factors, stromal COL11A1 provided better patient risk stratification. DCIS with higher stromal COL11A1 expression showed poor outcome even with adjuvant radiotherapy management. In conclusion, overexpression of stromal COL11A1 is associated with invasive recurrence in DCIS and is a potential marker to predict the response to radiotherapy.

A key genomic subtype associated with lymphovascular invasion in invasive breast cancer.

BACKGROUND: Lymphovascular invasion (LVI) is associated with the development of metastasis in invasive breast cancer (BC). However, the complex molecular mechanisms of LVI, which overlap with other oncogenic pathways, remain unclear. This study, using available large transcriptomic datasets, aims to identify genes associated with LVI in early-stage BC patients. METHODS: Gene expression data from the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) cohort (n = 1565) was used as a discovery dataset, and The Cancer Genome Atlas (TCGA; n = 854) cohort was used as a validation dataset. Key genes were identified on the basis of differential mRNA expression with respect to LVI status as characterised by histological review. The relationships among LVI-associated genomic subtype, clinicopathological features and patient outcomes were explored. RESULTS: A 99-gene set was identified that demonstrated significantly different expression between LVI-positive and LVI-negative cases. Clustering analysis with this gene set further divided cases into two molecular subtypes (subtypes 1 and 2), which were significantly associated with pathology-determined LVI status in both cohorts. The 10-year overall survival of subtype 2 was significantly worse than that of subtype 1. CONCLUSION: This study demonstrates that LVI in BC is associated with a specific transcriptomic profile with potential prognostic value.