Exploring molecular portraits defining lymphovascular invasion in invasive breast cancer patients

BACKGROUND: Lymphovascular invasion (LVI) is a robust clinicopathological parameter of poor prognosis in invasive breast cancer (BC). The accurate detection of tumour emboli within lymphovascular cavities in invasive BC can predict the unfavourable outcome for BC patients, including distant metastasis, recurrence, and short survival. The molecular drivers of LVI in invasive BC patients remain poorly defined. We sought to determine if specific transcriptomic and proteomic profiles regulate the LVI mechanisms in invasive BC. METHODOLOGIES: Meticulous detection of LVI occurrence was performed with histological and immunohistochemical staining on invasive BC full-face tissue sections to construct the LVI-supervised (LVI+/-) cohort, (n=170). All LVI- patients in this cohort were lymph node negative (LN-). The extraction of total RNA from the LVI+/- specimens followed by RNA microarray experiment and data analysis, utilising the linear models for microarray data (LIMMA) package for differential expression, yielded a large cluster of differentially expressed genes (DEG) associated with LVI, (n=1154). The last DEGs were validated against an external cohort of invasive BC patients under the same experimental conditions. We aimed to focus and prioritise our selection of LVI candidates through the statistical assessment of copy number aberrations (CNA) focusing on the top DEGs, (n=100), associated significantly with LVI according to the LIMMA analysis. CNA gain ratios were also calculated for each DEG in the LVI+ and LVI- categories. To avoid the dismissing of crucial LVI-drivers, an exhaustive and investigative approach was also applied and included all of the 1154 DEGs to identify the upregulated gene signatures associated with the LVI+ and LVI- cases in the LVI+/- cohort. Clinicopathological relevance and LVI associations were assessed for the identified LVI-DEGs in the METABRIC cohort, (n=1980), a subset of METABRIC cohort, including LVI-/LN+/- cases, (n=296), and the LVI+/- cohort, (n=170). Fewer LVI-candidates were selected for protein studies. Immunohistochemistry (IHC) protocols were performed to assess the altered protein expression for the qualified LVI-DEGs on tissue microarray (TMA) sections derived from the Nottingham primary series (NPS) of invasive BC, (n=2497). The following experiment aimed to identify the LVI differentially expressed proteins (DEP) utilising high-performance liquid chromatography/mass spectrometry (HPLC/MS) as a high-throughput screening technique. An LVI-supervised cohort consisted of invasive BC luminal-A phenotype was constructed, (n=80). The LVI status for these patients was defined histologically and immunohistochemically, and it comprised LVI+, (n=41), and LVI-, (n=39) cases. These specimens were used for total protein extraction, digestion, purification, and injected in Eksigent ekspert nano LC 425 system, then, analysed on a Sciex TripleTOF 6600 mass spectrometer. RESULTS: The CNA/CNA gain ratios analyses revealed a promising cluster of LVI-DEGs, (n=12). These LVI-DEGs are: MCFD2 (p-value < 0.02, CNA-gain = 4.56); CCT8 (p-value < 0.03, CNA-gain = 2.73); CUL4A (p-value < 0.002, CNA-gain = 1.59); ASCC3 (p-value < 0.01, CNA-gain = 1.36); DDX59 (p-value < 0.0001, CNA-gain = 1.11); ARFGEF1 (p-value < 1.0E-9, CNA-gain = 1.07); CD46 (p-value < 0.01, CNA-gain = 1.03); HRSP12 (p-value < 0.00008, CNA-gain = 0.91); MED23 (p-value < 0.008, CNA-gain = 0.91); EDEM (p-value < 0.01, CNA-gain = 0.86); FAM103A1 (p-value < 0.007, CNA-gain = 0.30); and PPP2CB (p-value < 0.0007, CNA-gain = 0.18). Exploratory bioinformatics search identified that SNPA23 is a co-expressed gene with MCFD2 and CCT8, the top LVI-candidates in the CNA analysis. The associations between MCFD2, CCT8, and SNAP23 against LVI were interrogated on the protein level with IHC. The exhaustive transcriptomic/LVI analyses has also discovered two unique clusters of LVI-DEGs. The first cluster included upregulated transcripts in the LVI+ category, (UP-13/LVI+). These transcripts are: PPM1G (p-value < 0.0001), SEC14L1 (p-value < 0.005), P4HA2 (p-value < 0.007), CTSD (p-value < 0.009), ITPRIP (p-value < 0.016), ZDHHC9 (p-value < 0.031), PILRA (p-value < 0.031), PDLIM7 (p-value < 0.032), RNASE1 (p-value < 0.032), SLC3A2 (p-value < 0.032), IKZF3 (p-value < 0.036), SIGLEC1 (p-value < 0.039), and SLC6A9 (p-value < 0.047). The second cluster included upregulated transcripts in the LVI- category, (UP-22/LVI-). These transcripts are: LPAR1 (p-value < 0.0002), CXCL12 (p-value < 0.001), ELP5 (p-value < 0.002), ECM2 (p-value < 0.004), APCDD1 (p-value < 0.005), FBLN1 (p-value < 0.005), ANXA5 (p-value < 0.008), INKA1 (p-value < 0.011), TMEM98 (p-value < 0.012), FEZ1 (p-value < 0.016), THAP4 (p-value < 0.016), TNFRSF19 (p-value < 0.016), NBR1 (p-value < 0.018), WISP2 (p-value < 0.019), CNRIP (p-value < 0.020), ITSN1 (p-value < 0.021), C1S (p-value < 0.022), TNC (p-value < 0.028), PRRX1 (p-value < 0.030), STAT3 (p-value < 0.035), TSC1 (p-value < 0.036), and TWIST2 (p-value < 0.047). The association between SEC14L1 and LVI was investigated on the protein level with IHC. The cytosolic protein expressions of MCFD2 and SEC14L1, the nuclear protein expression of CCT8, and the membranous protein expression of SNAP23 illustrated strong associations with LVI in invasive BC. Cytosolic MCFD2 and nuclear CCT8 exhibited qualities of LVI inhibitors, (p-value < 0.003) and (p-value < 0.010) respectively. On the other hand, cytosolic SEC14L1 and membranous SNAP23 displayed strong associations with actual LVI occurrences, (p-value < 0.0003) and (p-value < 0.020) correspondingly. The HPLC/MS differential protein expression identified 370 DEP associated with LVI. Among these DEPs, we identified 56 DEP overlapped with the identifiers of DEGs produced by the LVI/RNA microarray experiment. These DEPs are: ACTB, ACTG1, ANP32A, ARHGDIA, ARPC5, CCT8, COL6A2, COMT, DBI, DDX17, DYNLRB1, EIF4E, ERH, FBLN1, H2AFY, HMGB2, HNRNPA2B1, HNRNPH3, HNRNPK, HSP90AA1, HSPA4, ILF3, KCTD12, LGALS3, LMNA, OLA1, PGAM1, PPIA, PRRC1, PSMA6, PTGES3, PTMA, RAC1, RBBP4, RPL10A, RPL7, RPL9, RPLP1, RPS11, RPS18, RPS2, RPS28, RPS4X, RPS7, RPSA, SAR1A, SEC61B, SNRPE, SRSF3, SUB1, TPI1, TUBB6, UFM1, UPF1, VDAC2, and YWHAE. As the flow of LVI-triggering genetic codes should be transcripted first into mRNA then translated into functional proteins, we utilised the METABRIC transcriptomic data and applied Mann-Whitney test for median comparison to confirm the correlations between LVI and the 56 LVI-DEP on the mRNA level. We identified a cluster of DEGs/DEPs according to the RNA microarray and HPLC/MS experiments. These LVI- DEGs/DEPs are: HSP90AA1 (p-value < 0.0001), ARHGDIA (p-value < 0.004), FBLN1 (p-value < 0.005), DYNLRB1 (p-value < 0.018), VDAC2 (p-value < 0.030), RPS18 (p-value < 0.037), RPL10A (p-value < 0.046), and PPIA (p-value < 0.047). CONCLUSION: To conclude, the LVI initiation or repression in invasive BC may be attributed to altered expression of a cluster of genes and proteins. On the transcriptomic level, the LVI definite incidences were associated with the UP-13/LVI+ cluster of mRNAs that were preferentially upregulated in the luminal-B, HER2-enriched, and basal-like intrinsic subtypes. The proteins encoded by these mRNAs are involved in several pathways including intracellular protein trafficking, adaptive immune system, signalling by nuclear receptors, transmembrane glycoprotein interactions, extracellular matrix organisation and degradation, Na+/Cl- dependent neurotransmitter transporters, SLC-mediated transmembrane transport. In contrast, the absence of LVI was associated with a different cluster of mRNAs which were overexpressed in the vast majority of normal-like and luminal-A tumours. This explorative study of LVI provided novel LVI-signature in invasive BC with relevant prognostic utilities. In the future, elaborated and well-designed functional studies may reveal better insights on the contributions for each identified LVI-candidate in driving or inhibiting LVI mechanisms in invasive BC patients

Chemokine (C-C motif) receptor 7 (CCR7) associates with the tumour immune microenvironment but not progression in invasive breast carcinoma

Some previous studies have reported that the chemokine (C-C motif) receptor 7 (CCR7) plays a role in breast cancer, is associated with lymph node metastasis and drives the site of distant metastasis. However, the impact of its expression on patient outcome and its association with tumour infiltrating inflammatory cells remain to be validated. We evaluated CCR7 protein expression by immunohistochemistry in a large well characterized cohort (n = 866) of early invasive primary breast cancers. CCR7 was expressed in the cytoplasm and membrane of tumour cells. We observed a weak positive association of high CCR7 expression when in either cellular component, but not both together, with axillary lymph node stage 3 tumours (p = 0.043). Logistic regression analysis of lymph node stage revealed no independent predictive value for CCR7 expression. CCR7 expression was higher in HER2 positive tumours (p = 0.03) and associated with positive CD68+ FOXP3+ tumour infiltrating cells. CCR7 staining was negatively associated with CD3+ cells. There was no significant association of CCR7 expression with breast cancer recurrence or survival. We conclude that while CCR7 is not a useful biomarker for predicting lymph node metastasis, it may reflect altered intra- and inter-cellular signalling related to the immune microenvironment. The subcellular localization of CCR7 appears to affect the nature of these interactions

Ki67 expression in invasive breast cancer: the use of tissue microarrays compared with whole tissue sections.

BACKGROUND: Although the prognostic value of Ki67 in breast cancer is well documented, using optimal cut-points for patient stratification, reproducibility of the scoring and interpretation of the results remains a matter of debate particularly when using tissue microarrays (TMAs). This study aims to assess Ki67 expression assessed on TMAs and their matched whole tissue sections (WTS). Moreover, whether the cut-off used for WTS is reproducible on TMA in BC molecular classes and the association between Ki67 expression cut-off, assessed on TMAs and WTS, and clinicopathological parameters and patient outcome were tested. METHOD: A large series (n = 707) of primary invasive breast tumours were immunostained for Ki67 using both TMA and WTS and assessed as percentage staining and correlated with each other, clinicopathological parameters and patient outcome. In addition, MKI67 mRNA expression was correlated with Ki67 protein levels on WTS and TMAs in a subset of cases included in the METABRIC study. RESULTS: There was moderate concordance in Ki67 expression between WTS and TMA when analysed as a continuous variable (Intraclass correlation coefficient = 0.61) and low concordance when dichotomised (kappa value = 0.3). TMA showed low levels of Ki67 with mean percentage of expression of 35 and 22% on WTS and TMA, respectively. MKI67 mRNA expression was significantly correlated with protein expression determined on WTS (Spearman Correlation, r = 0.52) and to a lesser extent on TMA (r = 0.34) (p < 0.001). Regarding prediction of patient outcome, statistically significant differences were detected upon stratification of patients with tumours expressing Ki67 at 10, 15, 20, 25 or 30% in TMA. Using TMA, ≥20% Ki67 provided the best prognostic cut-off particularly in triple-negative and HER2-positive classes. CONCLUSION: Ki67 expression in breast cancer can be evaluated using TMA although different cut-points are required to emulate results from WTS. A cut-off of ≥20% for Ki67 expression in BC provides the best prognostic correlations when TMAs are used

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 (less than 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

Utility of Ankyrin 3 as a Prognostic Marker in Androgen-Receptor-Positive Breast Cancer Running Title: Prognostic Value of ANK3 in AR-Positive Breast Cancer

© 2019, Springer Science+Business Media, LLC, part of Springer Nature. 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

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

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-BRCA 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, TGFbeta1 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

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-BRCA 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, TGFbeta1 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 signifcance 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 signifcantly 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 signifcantly 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 identifed a subgroup of BC with aggressive behaviour and provided independent prognostic information