SHON expression predicts response and relapse risk of breast cancer patients after anthracycline-based combination chemotherapy or tamoxifen treatment.

BACKGROUND: SHON nuclear expression (SHON-Nuc+) was previously reported to predict clinical outcomes to tamoxifen therapy in ERα+ breast cancer (BC). Herein we determined if SHON expression detected by specific monoclonal antibodies could provide a more accurate prediction and serve as a biomarker for anthracycline-based combination chemotherapy (ACT). METHODS: SHON expression was determined by immunohistochemistry in the Nottingham early-stage-BC cohort (n = 1,650) who, if eligible, received adjuvant tamoxifen; the Nottingham ERα- early-stage-BC (n = 697) patients who received adjuvant ACT; and the Nottingham locally advanced-BC cohort who received pre-operative ACT with/without taxanes (Neo-ACT, n = 120) and if eligible, 5-year adjuvant tamoxifen treatment. Prognostic significance of SHON and its relationship with the clinical outcome of treatments were analysed. RESULTS: As previously reported, SHON-Nuc+ in high risk/ERα+ patients was significantly associated with a 48% death risk reduction after exclusive adjuvant tamoxifen treatment compared with SHON-Nuc- [HR (95% CI) = 0.52 (0.34-0.78), p = 0.002]. Meanwhile, in ERα- patients treated with adjuvant ACT, SHON cytoplasmic expression (SHON-Cyto+) was significantly associated with a 50% death risk reduction compared with SHON-Cyto- [HR (95% CI) = 0.50 (0.34-0.73), p = 0.0003]. Moreover, in patients received Neo-ACT, SHON-Nuc- or SHON-Cyto+ was associated with an increased pathological complete response (pCR) compared with SHON-Nuc+ [21 vs 4%; OR (95% CI) = 5.88 (1.28-27.03), p = 0.012], or SHON-Cyto- [20.5 vs. 4.5%; OR (95% CI) = 5.43 (1.18-25.03), p = 0.017], respectively. After receiving Neo-ACT, patients with SHON-Nuc+ had a significantly lower distant relapse risk compared to those with SHON-Nuc- [HR (95% CI) = 0.41 (0.19-0.87), p = 0.038], whereas SHON-Cyto+ patients had a significantly higher distant relapse risk compared to SHON-Cyto- patients [HR (95% CI) = 4.63 (1.05-20.39), p = 0.043]. Furthermore, multivariate Cox regression analyses revealed that SHON-Cyto+ was independently associated with a higher risk of distant relapse after Neo-ACT and 5-year tamoxifen treatment [HR (95% CI) = 5.08 (1.13-44.52), p = 0.037]. The interaction term between ERα status and SHON-Nuc+ (p = 0.005), and between SHON-Nuc+ and tamoxifen therapy (p = 0.007), were both statistically significant. CONCLUSION: SHON-Nuce+ in tumours predicts response to tamoxifen in ERα+ BC while SHON-Cyto+ predicts response to ACT.

Kinesin family member-18A (KIF18A) is a predictive biomarker of poor benefit from endocrine therapy in early ER+ breast cancer.

PURPOSE: Identification of effective and reliable biomarkers that could be used to predict the efficacy of endocrine therapy is of crucial importance to the management of oestrogen receptor positive (ER+) breast cancer (BC). KIF18A, a key regulator of cell cycle, is overexpressed in many human cancers, including BC. In this study, we investigated the role of KIF18A as a biomarker to predict the benefit from endocrine treatment in early ER + BC patients. METHODS: KIF18A expression was assessed at the genomic level using the METABRIC dataset to explore its prognostic and predictive value in ER + BC patients (n = 1506). Predictive significance of KIF18A mRNA was validated using KM-Plot datasets (n = 2061). KIF18A protein expression was assessed using immunohistochemistry in a large annotated series of early-stage ER + BC (n = 1592) with long-term follow-up. RESULTS: High mRNA and protein expression of KIF18A were associated with short recurrence-free survival (RFS), distant-metastasis free survival (DMFS) and BC specific survival (all P < 0.05) in ER + BC in patients who received no adjuvant treatment or adjuvant endocrine therapy. In multivariate analysis, high KIF18A expression was an independent prognostic biomarker for poor RFS (P = 0.027) and DMFS (P = 0.028) in patients treated with adjuvant endocrine therapy. CONCLUSION: KIF18A appears to be a candidate biomarker of a subgroup of ER + BC characterised by poor clinical outcome. High KIF18A expression has prognostic significance to predict poor benefit from endocrine treatment for patients with ER + BC. Therefore, measurement of KIF18A on ER + BC patients prior to treatment could guide clinician decision on benefit from endocrine therapy.

The combined expression of solute carriers is associated with a poor prognosis in highly proliferative ER+ breast cancer.

PURPOSE: Breast cancer (BC) is a heterogeneous disease characterised by variant biology, metabolic activity, and patient outcome. Glutamine availability for growth and progression of BC is important in several BC subtypes. This study aimed to evaluate the biological and prognostic role of the combined expression of key glutamine transporters, SLC1A5, SLC7A5, and SLC3A2 in BC with emphasis on the intrinsic molecular subtypes. METHODS: SLC1A5, SLC7A5, and SLC3A2 were assessed at the protein level, using immunohistochemistry on tissue microarrays constructed from a large well-characterised BC cohort (n = 2248). Patients were stratified into accredited clusters based on protein expression and correlated with clinicopathological parameters, molecular subtypes, and patient outcome. RESULTS: Clustering analysis of SLC1A5, SLC7A5, and SLC3A2 identified three clusters low SLCs (SLC1A5-/SLC7A5-/SLC3A2-), high SLC1A5 (SLC1A5+/SLC7A5-/SLC3A2-), and high SLCs (SLC1A5+/SLC7A5+/SLC3A2+) which had distinct correlations to known prognostic factors and patient outcome (p < 0.001). The key regulator of tumour cell metabolism, c-MYC, was significantly expressed in tumours in the high SLC cluster (p < 0.001). When different BC subtypes were considered, the association with the poor outcome was observed in the ER+ high proliferation/luminal B class only (p = 0.003). In multivariate analysis, SLC clusters were independent risk factor for shorter BC-specific survival (p = 0.001). CONCLUSION: The co-operative expression of SLC1A5, SLC7A5, and SLC3A2 appears to play a role in the aggressive subclass of ER+ high proliferation/luminal BC, driven by c-MYC, and therefore have the potential to act as therapeutic targets, particularly in synergism.

Glutamate dehydrogenase (GLUD1) expression in breast cancer.

BACKGROUND: Dysregulated cellular metabolism is one of the hallmarks of cancer with some tumours utilising the glutamine metabolism pathway for their sustained proliferation and survival. Glutamate dehydrogenase (GLUD1) is a key enzyme in glutaminolysis converting glutamate to α-ketoglutarate for entry into the TCA cycle. Breast cancer (BC) comprises a heterogeneous group of tumours in terms of molecular biology and clinical behaviour, and we have previously shown that altered glutamine metabolism varies substantially among the different molecular subtypes. We hypothesise that the prognostic value of GLUD1 expression will differ between the BC molecular subtypes and may act as a potential therapeutic target for BC tumours. METHODS: GLUD1 was assessed at the DNA, mRNA (n = 1980) and protein (n = 1300) levels in large, well-characterised cohorts and correlated with clinicopathological parameters, molecular subtypes, patient outcome, and treatments. RESULTS: There was a correlation between GLUD1 mRNA and GLUD1 protein expression which were highly expressed in low grade luminal/ER + BC (p < 0.01). GLUD1 mRNA and protein was associated with good patient outcome but not in any specific molecular subtypes. However, high GLUD1 protein expression was associated with a better outcome in triple negative (TN) patients treated with chemotherapy (p = 0.03). High GLUD1 mRNA was associated with the glutamine transporter, SLC1A5, and leucine transporter, SLC7A8 as well as mTOR (p < 0.0001). CONCLUSION: We provide comprehensive data indicating GLUD1 plays an important role in luminal/ER + BC. GLUD1 expression predicts a better patient outcome and we show that it has the potential for predicting response to chemotherapy in TNBC patients.

Utility of ankyrin 3 as a prognostic marker in androgen-receptor-positive breast cancer.

PURPOSE: Androgen receptor (AR) and AR signaling pathways are thought to play a role in breast cancer (BC) and are potentially related to treatment responses and outcomes. Ankyrin 3 (ANK3) is associated with AR stability in cancer cells. In the present study, we investigated the clinicopathological utility of ANK3 expression with emphasis on AR and its associated signalling pathway at transcriptomic and proteomic phases. PATIENTS AND METHODS: The Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) cohort (n = 1980) and The Cancer Genome Atlas (TCGA) dataset (n = 1039) were used to assess the expression and significance of ANK3 mRNA and other AR signalling pathway-associated gene signature. Using immunohistochemistry, ANK3 protein expression was evaluated in large (n = 982) cohort of early-stage BC with long-term follow-up and compared with clinicopathological characteristics and its prognostic value in the whole cohort and the subgroups stratified by AR protein expression. RESULTS: An AR-related gene signature was developed, comprising 20 genes, which included ANK3. This AR-related gene signature was significantly associated with AR mRNA expression, oestrogen receptor, human epidermal growth factor receptor 2 (HER2) status and the patients' outcomes. In tumours with high AR protein expression (n = 614), high ANK3 protein expression was significantly associated with progesterone receptor positivity and it was independently associated with the good outcomes (p = 0.025). CONCLUSIONS: This study indicates that ANK3 is related to AR signalling pathway and is associated with BC prognosis.

The amino acid transporter SLC7A5 confers a poor prognosis in the highly proliferative breast cancer subtypes and is a key therapeutic target in luminal B tumours.

BACKGROUND: Breast cancer (BC) is a heterogeneous disease characterised by variant biology and patient outcome. The amino acid transporter, SLC7A5, plays a role in BC although its impact on patient outcome in different BC subtypes remains to be validated. This study aimed to determine whether the clinicopathological and prognostic value of SLC7A5 is different within the molecular classes of BC. METHODS: SLC7A5 was assessed at the genomic level, using Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) data (n = 1980), and proteomic level, using immunohistochemical analysis and tissue microarray (TMA) (n = 2664; 1110 training and 1554 validation sets) in well-characterised primary BC cohorts. SLC7A5 expression correlated with clinicopathological and biological parameters, molecular subtypes and patient outcome. RESULTS: SLC7A5 mRNA and protein expression were strongly correlated with larger tumour size and higher grade. High expression was observed in triple negative (TN), human epidermal growth factor receptor 2 (HER2)+, and luminal B subtypes. SLC7A5 mRNA and protein expression was significantly associated with the expression of the key regulator of tumour cell metabolism, c-MYC, specifically in luminal B tumours only (p = 0.001). High expression of SLC7A5 mRNA and protein was associated with poor patient outcome (p < 0.001) but only in the highly proliferative oestrogen receptor (ER)+/ luminal B (p = 0.007) and HER2+ classes of BC (p = 0.03). In multivariate analysis, SLC7A5 protein was an independent risk factor for shorter breast-cancer-specific survival only in ER+ high-proliferation tumours (p = 0.02). CONCLUSIONS: SLC7A5 appears to play a role in the aggressive highly proliferative ER+ subtype driven by MYC and could act as a potential therapeutic target. Functional assessment is necessary to reveal the specific role played by this transporter in the ER+ highly proliferative subclass and HER2+ subclass of BC.

The multifunctional solute carrier 3A2 (SLC3A2) confers a poor prognosis in the highly proliferative breast cancer subtypes.

Breast cancer (BC) is a heterogeneous disease characterised by variant biology, metabolic activity and patient outcome. This study aimed to evaluate the biological and prognostic value of the membrane solute carrier, SLC3A2 in BC with emphasis on the intrinsic molecular subtypes. SLC3A2 was assessed at the genomic level, using METABRIC data (n = 1980), and at the proteomic level, using immunohistochemistry on tissue microarray (TMA) sections constructed from a large well-characterised primary BC cohort (n = 2500). SLC3A2 expression was correlated with clinicopathological parameters, molecular subtypes and patient outcome. SLC3A2 mRNA and protein expression were strongly correlated with higher tumour grade and poor Nottingham prognostic index (NPI). High expression of SLC3A2 was observed in triple-negative (TN), HER2+ and ER+ high-proliferation subtypes. SLC3A2 mRNA and protein expression were significantly associated with the expression of c-MYC in all BC subtypes (p < 0.001). High expression of SLC3A2 protein was associated with poor patient outcome (p < 0.001), but only in the ER+ high-proliferation (p = 0.01) and TN (p = 0.04) subtypes. In multivariate analysis SLC3A2 protein was an independent risk factor for shorter BC-specific survival (p < 0.001). SLC3A2 appears to play a role in the aggressive BC subtypes driven by MYC and could act as a potential prognostic marker. Functional assessment is necessary to reveal its potential therapeutic value in the different BC subtypes.

MYC regulation of glutamine-proline regulatory axis is key in luminal B breast cancer.

BACKGROUND: Altered cellular metabolism is a hallmark of cancer and some are reliant on glutamine for sustained proliferation and survival. We hypothesise that the glutamine-proline regulatory axis has a key role in breast cancer (BC) in the highly proliferative classes. METHODS: Glutaminase (GLS), pyrroline-5-carboxylate synthetase (ALDH18A1), and pyrroline-5-carboxylate reductase 1 (PYCR1) were assessed at DNA/mRNA/protein levels in large, well-characterised cohorts. RESULTS: Gain of PYCR1 copy number and high PYCR1 mRNA was associated with Luminal B tumours. High ALDH18A1 and high GLS protein expression was observed in the oestrogen receptor (ER)+/human epidermal growth factor receptor (HER2)- high proliferation class (Luminal B) compared with ER+/HER2- low proliferation class (Luminal A) (P=0.030 and P=0.022 respectively), however this was not observed with mRNA. Cluster analysis of the glutamine-proline regulatory axis genes revealed significant associations with molecular subtypes of BC and patient outcome independent of standard clinicopathological parameters (P=0.012). High protein expression of the glutamine-proline enzymes were all associated with high MYC protein in Luminal B tumours only (P<0.001). CONCLUSIONS: We provide comprehensive clinical data indicating that the glutamine-proline regulatory axis plays an important role in the aggressive subclass of luminal BC and is therefore a potential therapeutic target.

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

BACKGROUND: Mediator 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. METHODS: MED7 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. RESULTS: High 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). CONCLUSIONS: MED7 is an important prognostic marker in BC, particularly in ER+luminal subtypes, associated with improved survival and warrants future functional analysis.

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

BACKGROUND: Ras 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. METHODS: The 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). RESULTS: In 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, TGFß1 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). CONCLUSIONS: High RAPH1 expression is correlated with aggressive breast cancer phenotypes and provides independent prognostic value in invasive breast cancer.

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

BACKGROUND: The 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. METHODS: The 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. RESULTS: KLHL7 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). CONCLUSIONS: KLHL7 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.

Thioredoxin-interacting protein is an independent risk stratifier for breast ductal carcinoma in situ.

Current clinicopathological parameters are useful predictors of breast ductal carcinoma in situ behavior, but they are insufficient to define high-risk patients for disease progression precisely. Thioredoxin-interacting protein (TXNIP) is a key player of oxidative stress. This study aims to evaluate the role of TXNIP as a predictor of ductal carcinoma in situ progression. Tissue microarrays from 776 pure ductal carcinoma in situ and 239 mixed ductal carcinoma in situ and invasive tumors were constructed. All patients were treated at a single institution with a long-term follow-up and TXNIP expression was assessed using immunohistochemistry. TXNIP expression was investigated in terms of associations with clinicopathological and molecular features and patient outcome. Loss/reduced cytoplasmic expression of TXNIP was associated with features of aggressiveness including high nuclear grade (p = 1.6 × 10-5), presence of comedo necrosis (p = 0.001), and estrogen receptor negative (ER-)/HER2- ductal carcinoma in situ (p = 4.6 × 10-5). Univariate analysis showed an inverse association between TXNIP expression and outcome in terms of shorter local recurrence-free survival (p = 0.009). Multivariable analyses showed that independent predictors of ductal carcinoma in situ recurrence were low TXNIP expression (p = 0.005, HR = 0.51, and 95% CI: 0.32-0.81), larger ductal carcinoma in situ size, and high nuclear grade. TXNIP functions as a tumor suppressor gene with loss of its expression associated with ductal carcinoma in situ recurrence. TXNIP can be used as a potentially useful marker in prognostic stratification of ductal carcinoma in situ for management decisions.

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 in breast cancer and its prognostic significance.

BACKGROUND AND AIMS: 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. METHODS AND RESULTS: Immunohistochemically defined immune cell subtypes, i.e. those expressing T-cell markers (CD3, CD8, and FOXP3), a B-cell marker (CD20) and a histiocytic marker (CD68), were evaluated in a large series (n = 1165) of invasive BCs. A subset of full-face haematoxylin and eosin (H&E)-stained slides were examined for TILs heterogeneity within primary tumours and the corresponding local recurrent carcinomas to investigate spatial and temporal TILs heterogeneity. H&E-stained sections from multiple tumour blocks (three or four blocks per case) representing different tumour areas were evaluated to assess TILs interslide heterogeneity and intraslide heterogeneity. Both average stromal TILs (AV-TILs) and hotspot stromal TILs (HS-TILs) were assessed. AV-TILs showed associations with all immune cell subtypes; however, CD3+ cells constituted the main component (mean number of CD3+ was 55), whereas CD20+ cells constituted the smallest component (mean number of CD20 was 13). There was no significant statistical difference between TILs across tumour blocks of the same case (P = 0.251 for AV-TILs and P = 0.162 for HS-TILs). Triple-negative breast cancer (TNBC) showed higher TILs percentage than other BC subtypes (P < 0.001). High AV-TILs, CD3+ cells, CD8+ cells and CD20+ cells were associated with longer survival in TNBC patients (P < 0.05). High AV-TILs in recurrent tumours showed a significant association with shorter post-recurrence survival (P = 0.004). CONCLUSIONS: Despite the heterogeneity of immune cell type components, average TILs in one full-face H&E-stained section reliably represent TILs in the whole tumour. TILs were associated with outcome in TNBC patients, as well as having prognostic significance for recurrent tumours.

Phenotypic characterisation of breast cancer: the role of CDC42.

PURPOSE: The molecular landscape of breast cancer (BC), especially of the Luminal A subtype, remains to be fully delineated. Transcriptomic data show that Luminal A tumours are enriched for aberrant expression of genes in the cell division control 42 homolog (CDC42) pathway. This study aims to investigate the protein expression of CDC42 in BC and assess its clinicopathological significance. METHODS: Expression of CDC42 protein was examined by immunohistochemistry on tissue microarrays in a well-characterised cohort of 895 early-stage (I-IIIa) primary invasive BCs. RESULTS: CDC42 expression was observed in both the cytoplasm and the nucleus of BC cells. High nuclear CDC42 expression demonstrated a significant correlation with ER-positive, low-grade tumours and was more common in the lobular histological subtype (all p < 0.001). In contrast, cytoplasmic CDC42 showed increased expression in the ductal subtype (p < 0.001) and correlated with negative prognostic features such as larger size, higher grade (p < 0.05) and higher Ki67 labelling index (p = 0.001). Nuclear CDC42 expression was associated with a longer BC-specific survival in all cases (p = 0.025) and in luminal ER-positive tumours (p = 0.011). In multivariate analyses including size, grade, lymph node stage and intrinsic subtype, CDC42 was an independent prognostic factor (p = 0.032). CONCLUSION: The results indicate that CDC42 is an important molecule in luminal BC, with prognostic significance.

Further evidence to support bimodality of oestrogen receptor expression in breast cancer.

AIMS: Although oestrogen receptor (ER)-negative breast cancers (BCs) do not respond to hormone therapy, the response of ER-positive BCs is reported to be variable, which may suggest a dose-dependent effect. The aim of this study was to assess the pattern of ER expression in BCs at the protein (immunohistochemistry) and transcriptome (microarray-based gene expression) levels. METHODS AND RESULTS: ER immunohistochemical (IHC) expression was assessed in a large series of BCs, including 3649 core biopsies and 1892 cases prepared as tissue microarrays (TMAs) stained with specific antibodies. ESR1 mRNA expression was assessed in the METABRIC study (1980 cases), by the use of the Linear Models for Microarray Data (limma) software, and the results were compared with protein levels. IHC data confirmed the bimodality of ER expression, with 92.2% and 89.2% of the cases showing completely negative (<1%) or highly positive (≥70%) expression on the cores and TMAs, respectively. Weakly positive cases (1-10%) and intermediately positive (11-69%) cases were infrequent (2.7% and 5.1%, and 1.6% and 9.2%, in cores and TMAs, respectively), and did not show survival difference from ER-negative tumours. When full-face sections of the corresponding excision specimens were immunostained, 47% of the ER-low/intermediate group were deemed to be ER-negative. Transcriptomic data not only showed a significant correlation between ESR1 mRNA and protein expression levels, but also confirmed the bimodality of ER expression at the mRNA level. CONCLUSIONS: Our study provides further evidence that ER expression is bimodal, and that it is observed at both the mRNA and protein levels. The reported poor survival of BC patients with low ER expression in the early clinical trials may be related to the inclusion of ER-negative cases.

Prognostic significance of tumour infiltrating B lymphocytes in breast ductal carcinoma in situ.

AIMS: Tumour-infiltrating lymphocytes (TILs) are an important component of the immune response to cancer and have a prognostic value in breast cancer. Although several studies have investigated the role of T lymphocytes in breast cancer, the role of B lymphocytes (TIL-Bs) in ductal carcinoma in situ (DCIS) remains uncertain. This study aimed to assess the role of TIL-Bs in DCIS. METHODS AND RESULTS: Eighty DCIS cases (36 pure DCIS and 44 mixed with invasive cancer) were stained immunohistochemically for B lineage markers CD19, CD20 and the plasma cell marker CD138. TIL-Bs density and localization were assessed, including relation to the in-situ and invasive components. An association with clinicopathological data and patient outcome was performed. Pure DCIS showed a higher number of TIL-Bs and lymphoid aggregates than DCIS associated with invasion. In pure DCIS, a higher number of peri- and paratumoral TIL-Bs was associated significantly with large tumour size (P = 0.016), hormone receptor (ER/PR) negative (P = 0.008) and HER2+ status (P = 0.010). In tumours with mixed DCIS and invasive components, cases with high-density B lymphocytes, irrespective of their location or topographic distribution, were associated significantly with variables of poor prognosis, including larger size, high grade, lymphovascular invasion, lymph node metastases, ER/PR-negative and HER2+ status. Outcome analysis showed that pure DCIS associated with higher numbers of B lymphocytes had shorter recurrence-free interval (P = 0.04); however, the association was not significant with the CD138+ plasma cell count (P = 0.07). CONCLUSION: Assessment of TIL-B cells based on location and topographic distribution can provide prognostic information. Validation in a larger cohort is warranted.

MYC functions are specific in biological subtypes of breast cancer and confers resistance to endocrine therapy in luminal tumours.

BACKGROUND: MYC is amplified in approximately 15% of breast cancers (BCs) and is associated with poor outcome. c-MYC protein is multi-faceted and participates in many aspects of cellular function and is linked with therapeutic response in BCs. We hypothesised that the functional role of c-MYC differs between molecular subtypes of BCs. METHODS: We therefore investigated the correlation between c-MYC protein expression and other proteins involved in different cellular functions together with clinicopathological parameters, patients' outcome and treatments in a large early-stage molecularly characterised series of primary invasive BCs (n=1106) using immunohistochemistry. The METABRIC BC cohort (n=1980) was evaluated for MYC mRNA expression and a systems biology approach utilised to identify genes associated with MYC in the different BC molecular subtypes. RESULTS: High MYC and c-MYC expression was significantly associated with poor prognostic factors, including grade and basal-like BCs. In luminal A tumours, c-MYC was associated with ATM (P=0.005), Cyclin B1 (P=0.002), PIK3CA (P=0.009) and Ki67 (P<0.001). In contrast, in basal-like tumours, c-MYC showed positive association with Cyclin E (P=0.003) and p16 (P=0.042) expression only. c-MYC was an independent predictor of a shorter distant metastases-free survival in luminal A LN+ tumours treated with endocrine therapy (ET; P=0.013). In luminal tumours treated with ET, MYC mRNA expression was associated with BC-specific survival (P=0.001). In ER-positive tumours, MYC was associated with expression of translational genes while in ER-negative tumours it was associated with upregulation of glucose metabolism genes. CONCLUSIONS: c-MYC function is associated with specific molecular subtypes of BCs and its overexpression confers resistance to ET. The diverse mechanisms of c-MYC function in the different molecular classes of BCs warrants further investigation particularly as potential therapeutic targets.

Clinical utility of reverse phase protein array for molecular classification of breast cancer.

Reverse Phase Protein Array (RPPA) represents a sensitive and high-throughput technique allowing simultaneous quantitation of protein expression levels in biological samples. This study aimed to confirm the ability of RPPA to classify archival formalin-fixed paraffin-embedded (FFPE) breast cancer tissues into molecular classes used in the Nottingham prognostic index plus (NPI+) determined by immunohistochemistry (IHC). Proteins were extracted from FFPE breast cancer tissues using three extraction protocols: the Q-proteome FFPE Tissue Kit (Qiagen, Hilden, Germany) and two in-house methods using Laemmli buffer with either incubation for 20 min or 2 h at 105 °C. Two preparation methods, full-face sections and macrodissection, were used to assess the yield and quality of protein extracts. Ten biomarkers used for the NPI+ (ER, PgR, HER2, Cytokeratins 5/6 and 7/8, EGFR, HER3, HER4, p53 and Mucin 1) were quantified using RPPA and compared to results determined by IHC. The Q-proteome FFPE Tissue Kit produced significantly higher protein concentration and signal intensities. The intra- and inter-array reproducibility assessment indicated that RPPA using FFPE lysates was a highly reproducible and robust technique. Expression of the biomarkers individually and in combination using RPPA was highly consistent with IHC results. Macrodissection of the invasive tumour component gave more reliable results with the majority of biomarkers determined by IHC, (80 % concordance) compared with full-face sections (60 % concordance). Our results provide evidence for the technical feasibility of RPPA for high-throughput protein expression profiling of FFPE breast cancer tissues. The sensitivity of the technique is related to the quality of extracted protein and purity of tumour tissue. RPPA could provide a quantitative technique alternative to IHC for the biomarkers used in the NPI+.

The prognostic significance of STAT3 in invasive breast cancer: analysis of protein and mRNA expressions in large cohorts.

Signal transducer and activator of transcription (STAT) transcription factors family are involved in diverse cellular biological functions. Reports regarding the prognostic impact of STAT3 expression in breast cancer (BC) are variable whether being a factor of poor or good prognosis. Immunohistochemical expression of phospho-STAT3 (pSTAT3) was studied in large series of invasive BC (n = 1270). pSTAT3 and STAT3 were quantified using reverse phase protein array (RPPA) on proteins extracted from macro-dissected FFPE tissues (n = 49 cases). STAT3 gene expression in the METABRIC cohort was also investigated. STAT3 gene expression prognostic impact was externally validated using the online BC gene expression data (n = 26 datasets, 4.177 patients). pSTAT3 was expressed in the nuclei and cytoplasm of invasive BC cells. Nuclear pSTAT3 overexpression was positively associated with smaller tumour size, lower grade, good NPI, negative lymphovascular invasion (LVI), ER+, PgR+, p53-, HER2-, and low Ki67LI and an improved breast cancer-specific survival (BCSS), independently of other factors. On RPPA, the mean pSTAT3 and STAT3 expressions were higher in ER+, PgR+, and smaller size tumours. Higher STAT3 transcripts in the METABRIC cohort were observed in cases with favourable prognostic criteria and as well as improved BCSS within the whole cohort, ER+ cohort with and without hormonal therapy, and ER- cohort including those who did not receive adjuvant chemotherapy. Pooled STAT3 gene expression data in the external validation cohort showed an association with improved patients' outcome (P < 0.001, HR = 0.84, 95 % CI 0.79-0.90). Results of this study suggest nuclear localisation of pSTAT3 as favourable prognostic marker in invasive BC, results re-enforced by analysis of STAT3 gene expression data. This good prognostic advantage was maintained in patients who received and who did not receive adjuvant therapy. Therefore, STAT3 could have context-dependent molecular roles of in BC, results which warrant further prospective verification in clinical trials.