Glycan gene expression signatures in normal and malignant breast tissue; possible role in diagnosis and progression

Glycosylation is the stepwise procedure of covalent attachment of oligosaccharide chains to proteins or lipids, and alterations in this process have been associated with malignant transformation. Simultaneous analysis of the expression of all glycan-related genes clearly gives the advantage of enabling a comprehensive view of the genetic background of the glycobiological changes in cancer cells. Studies focusing on the expression of the whole glycome have now become possible, which prompted us to review the present knowledge on glycosylation in relation to breast cancer diagnosis and progression, in the light of available expression data from tumors and breast tissue of healthy individuals. We used various data resources to select a set of 419 functionally relevant genes involved in synthesis, degradation and binding of N-linked and O-linked glycans, Lewis antigens, glycosaminoglycans (chondroitin, heparin and keratan sulfate in addition to hyaluronan) and glycosphingolipids. Such glycans are involved in a number of processes relevant to carcinogenesis, including regulation of growth factors/growth factor receptors, cell–cell adhesion and motility as well as immune system modulation. Expression analysis of these glycan-related genes revealed that mRNA levels for many of them differ significantly between normal and malignant breast tissue. An associative analysis of these genes in the context of current knowledge of their function in protein glycosylation and connection(s) to cancer indicated that synthesis, degradation and adhesion mediated by glycans may be altered drastically in mammary carcinomas. Although further analysis is needed to assess how changes in mRNA levels of glycan genes influence a cell's glycome and the precise role that such altered glycan structures play in the pathogenesis of the disease, lessons drawn from this study may help in determining directions for future research in the rapidly-developing field of glycobiology.     

Validation of the prognostic gene portfolio,ClinicoMolecular Triad Classification,using an independent prospective breast cancer cohort and external patient populations

Introduction

Using genome-wide expression profiles of a prospective training cohort of breast cancer patients, ClinicoMolecular Triad Classification (CMTC) was recently developed to classify breast cancers into three clinically relevant groups to aid treatment decisions. CMTC was found to be both prognostic and predictive in a large external breast cancer cohort in that study. This study serves to validate the reproducibility of CMTC and its prognostic value using independent patient cohorts.

Methods

An independent internal cohort (n = 284) and a new external cohort (n = 2,181) were used to validate the association of CMTC between clinicopathological factors, 12 known gene signatures, two molecular subtype classifiers, and 19 oncogenic signalling pathway activities, and to reproduce the abilities of CMTC to predict clinical outcomes of breast cancer. In addition, we also updated the outcome data of the original training cohort (n = 147).

Results

The original training cohort reached a statistically significant difference (p < 0.05) in disease-free survivals between the three CMTC groups after an additional two years of follow-up (median = 55 months). The prognostic value of the triad classification was reproduced in the second independent internal cohort and the new external validation cohort. CMTC achieved even higher prognostic significance when all available patients were analyzed (n = 4,851). Oncogenic pathways Myc, E2F1, Ras and β-catenin were again implicated in the high-risk groups.

Conclusions

Both prospective internal cohorts and the independent external cohorts reproduced the triad classification of CMTC and its prognostic significance. CMTC is an independent prognostic predictor, and it outperformed 12 other known prognostic gene signatures, molecular subtype classifications, and all other standard prognostic clinicopathological factors. Our results support further development of CMTC portfolio into a guide for personalized breast cancer treatments.     

DNA methylation in serum of breast cancer patients: an independent prognostic marker

Changes in the status of DNA methylation are one of the most common molecular alterations in human neoplasia. Because it is possible to detect these epigenetic alterations in the bloodstream of patients, we investigated whether aberrant DNA methylation in patient pretherapeutic sera is of prognostic significance in breast cancer. Using MethyLight, a high-throughput DNA methylation assay, we analyzed 39 genes in a gene evaluation set, consisting of 10 sera from metastasized patients, 26 patients with primary breast cancer, and 10 control patients. To determine the prognostic value of genes identified within the gene evaluation set, we finally analyzed pretreatment sera of 24 patients having had no adjuvant treatment (training set) to determine their prognostic value. An independent test set consisting of 62 patients was then used to test the validity of genes and combinations of genes, which in the training set were found to be good prognostic markers. In the gene evaluation set we identified five genes (ESR1, APC, HSD17B4, HIC1, and RASSF1A). In the training set, patients with methylated serum DNA for RASSF1A and/or APC had the worst prognosis (P < 0.001). This finding was confirmed by analyzing serum samples from the independent test set (P = 0.007). When analyzing all 86 of the investigated patients, multivariate analysis showed methylated RASSF1A and/or APC serum DNA to be independently associated with poor outcome, with a relative risk for death of 5.7. DNA methylation of particular genes in pretherapeutic sera of breast cancer patients, especially of RASSF1A/APC, is more powerful than standard prognostic parameters.     

Breast cancer metastases are molecularly distinct from their primary tumors

Metastases have been widely thought to arise from rare, selected, mutation-bearing cells in the primary tumor. Recently, however, it has been proposed that breast tumors are imprinted ab initio with metastatic ability. Thus, there is a debate over whether 'phenotypic' disease progression is really associated with 'molecular' progression. We profiled 26 matched primary breast tumors and lymph node metastases and identified 270 probesets that could discriminate between the two categories. We then used an independent cohort of breast tumors (81 samples) and unmatched distant metastases (32 samples) to validate and refine this list down to a 126-probeset list. A representative subset of these genes was subjected to analysis by in situ hybridization, on a third independent cohort (57 primary breast tumors and matched lymph node metastases). This not only confirmed the expression profile data, but also allowed us to establish the cellular origin of the signals. One-third of the analysed representative genes (4 of 11) were expressed by the epithelial component. The four epithelial genes alone were able to discriminate primary breast tumors from their metastases. Finally, engineered alterations in the expression of two of the epithelial genes (SERPINB5 and LTF) modified cell motility in vitro, in accordance with a possible causal role in metastasis. Our results show that breast cancer metastases are molecularly distinct from their primary tumors.     

MIB-1 labelling index is an independent prognostic marker in primary breast cancer.

The proliferative activity of a tumour is considered to be an important prognostic factor in primary breast cancer. We have investigated the prognostic value of the MIB-1 labelling index in 341 patients with primary breast cancer and compared the results with the S-phase fraction in 220 patients of the same cohort. All patients were treated in one hospital and had a median follow-up of 128 months. No correlation between MIB-1 labelling and S-phase fraction could be demonstrated. MIB-1 had prognostic value for disease-free survival in the whole group of patients (P < 0.001) and in the node-negative subgroup (P < 0.001). In multivariate analysis, MIB-1 was an independent prognostic factor (P = 0.004) besides axillary lymph node status (P = 0.001). In univariate analysis high S-phase fraction was associated with decreased overall survival (P = 0.04); however, not in multivariate analysis. Moreover, S-phase fraction had a borderline prognostic significance for post-relapse survival in multivariate analysis (P= 0.08). Thus, in conclusion, the growth fraction of a tumour as determined by the MIB-1 labelling index is an important prognostic factor in patients with primary breast cancer.     

Hexokinase II in breast carcinoma: A potent prognostic factor associated with hypoxia‐inducible factor‐1α and Ki‐67

Hypoxia‐inducible factor‐1α (HIF‐1α) mediates adaptive responses to changes under tissue hypoxia in carcinoma cells by controlling the expression of various target genes. Previous studies have demonstrated that HIF‐1α is associated with adverse clinical outcome in breast carcinoma patients, but details of HIF‐1α's role have remained largely unknown. Therefore, in this study, we examined the expression profiles of HIF‐1α‐induced genes in 10 breast carcinoma cases using microarray data. As a result, we demonstrated that the status of hexokinase II (HKII) was associated with carcinoma recurrence in patients with these genes. The enzyme HKII is involved in the first, and rate‐limiting, step of glycolysis, but its clinical significance has not yet been examined in breast carcinoma. Therefore, we immunolocalized HKII in 118 breast carcinomas, and HKII immunoreactivity was detected in 44% of the cases. It is significantly associated with histological grade, Ki‐67 labeling index and HIF‐1α immunoreactivity. Also, HKII status is significantly associated with increased risk of recurrence and adverse clinical outcome in breast cancer patients. Subsequent multivariate analysis demonstrated that HKII status was an independent prognostic factor for disease‐free survival of patients. These results all suggest that HKII is induced by HIF‐1α and plays important roles in the proliferation and/or progression of breast carcinoma possibly through increased glycolytic activity. The status of HKII is therefore considered a potent prognostic factor in human breast cancer patients.     

EGR3 mRNA在乳腺癌组织中的表达及临床意义的生物信息学分析

目的:通过生物信息学方法分析早期生长反应因子3（early growth response 3,EGR3）在乳腺癌组织中的转录水平表达及其临床意义。方法:利用GEPIA数据库比较EGR3 mRNA在癌旁组织及乳腺癌组织中的表达差异;利用Linked Omics数据库比较不同分子分型及临床分期的乳腺癌组织中EGR3 mRNA的表达量,以明确EGR3 mRNA在乳腺癌中表达的临床意义;利用GEPIA数据库评价EGR3 mRNA表达对乳腺癌患者预后的影响,并采用Kaplan-Meier Plotter数据库的大规模验证队列对EGR3 mRNA的预后价值进行验证;最后利用Linked Omics数据库探索EGR3的共相关基因,将相关性最高的前50个基因生成在线热图。结果:在TCGA数据库及匹配了GTEx癌旁组织表达谱的两组队列中,EGR3 mRNA在乳腺癌组织中的表达量显著低于癌旁组织;在更高的T分期、M分期及临床TNM分期的乳腺癌组织中,EGR3 mRNA的表达量明显下调;此外,ER阴性、PR阴性的乳腺癌组织中,EGR3 mRNA的表达量也显著下调;通过分析GEPIA和Kaplan-Meier Plotter数据库的预后信息,表明EGR3 mRNA的低表达与更差的乳腺癌预后密切相关;最后,EGR3共表达基因分析结果分别显示了正、负相关性最高的前50个基因,初步探讨EGR3参与乳腺癌进程的潜在分子机制。结论:EGR3 mRNA在乳腺癌组织中低表达,且初步显示与乳腺癌的发生、发展及预后存在一定相关性,可以作为乳腺癌诊断、预后预测的潜在标志物。     

Pooled analysis of the prognostic relevance of progesterone receptor status in five German cohort studies

The progesterone receptor (PR) has been increasingly well described as an important mediator of the pathogenesis and progression of breast cancer. The aim of this study was to assess the role of PR status as a prognostic factor in addition to other well-established prognostic factors. Data from five independent German breast cancer centers were pooled. A total of 7,965 breast cancer patients were included for whom information about their PR status was known, as well as other patient and tumor characteristics commonly used as prognostic factors. Cox proportional hazards models were built to compare the predictive value of PR status in addition to age at diagnosis, tumor size, nodal status, grading, and estrogen receptor (ER) status. PR status significantly increased the accuracy of prognostic predictions with regard to overall survival, distant disease-free survival, and local recurrence-free survival. There were differences with regard to its prognostic value relative to subgroups such as nodal status, ER status, and grading. The prognostic value of PR status was greatest in patients with a positive nodal status, negative ER status, and low grading. The PR-status adds prognostic value in addition to ER status and should not be omitted from clinical routine testing. The significantly greater prognostic value in node-positive and high-grade tumors suggests a greater role in the progression of advanced and aggressive tumors.     

Autophagy-based survival prognosis in human colorectal carcinoma

The role of autophagy in cancers is controversial. Here we aim to determine the prognostic significance of autophagy in colorectal carcinoma patients, thereby allowing more rational development of therapeutic strategies. Through transmission electron microscopy, our data first demonstrated high frequency of defective mitochondria was strongly associated with poor overall survival in colorectal carcinoma. Next immunohistochemical study showed the expressions of Beclin 1, LC3B and Bcl-xL in both the center of tumor and adjacent noncancerous mucosal region were also correlated with overall survivals. We developed an autophagy signature for prognosis based on these three major autophagic proteins, further analysis suggested it was an independent prognostic biomarker and had its value even within single clinical stage. Combined TNM stage and this signature could significantly improve the accuracy of survival prognosis. To validate these immunohistochemical results, an internal testing cohort and an independent population were also included. Our findings suggest that autophagy plays an important role in the clinical cancer progression. Therefore autophagic proteins may be valuable prognostic biomarkers in the therapy of colorectal carcinoma and possibly other types of cancers.     

PD-1 Relevant Genes Predict the Prognosis of Breast Cancer and Their Prediction Effect in Tumor Response to Immunotherapy

Background: Immunotherapy is becoming a powerful approach in the treatment of breast cancer. However, high response rates in the majority of breast cancer patients have yet to be achieved. Materials and Methods: Based on public data sources, we identified 22 genes that are correlated with PD-1 expression as well as differentially expressed between tumor tissues and normal tissues, and high expression of all the key genes was correlated with a superior survival outcome. Using the least absolute shrinkage and selection operator Cox regression model, a risk score was constructed. Results: A multivariate Cox analysis showed that the constructed risk score was an independent prognostic factor (AUC of risk score: 0.63, HR of risk score: 11.7, C-index: 0.69). Furthermore, an analysis of TILs in the TCGA BRCA cohort identified 28 distinct immune cell types. T lymphocytes and myeloid-derived suppressor cells were enriched in low-risk patients. The risk score was positively correlated with immune checkpoint genes for PD-1, PD-L1, PD-L2, and CTLA4 (P < 0.05). In addition, two independent cohorts validated the prognostic value and the predictive value of the immunotherapy response of the risk score. Conclusions: The risk score demonstrated a stable predictive ability in breast cancer prognosis and a predictive value in tumor response to immunotherapy. The analysis of TILs revealed the correlation between risk score and immune cells, which also helps elucidate the complexity of the relationship between TILs and immunotherapy response.     

Prognostic significance of tumor/stromal caveolin-1 expression in breast cancer patients

Caveolin-1 (Cav-1) has been extensively characterized in cancer biological research. However, the role of Cav-1 in the interaction between tumor and stromal cells remains unclear. In the present study, we examined Cav-1 expression in tumor cells and stromal cells in breast cancer tissue by immunohistochemical analysis and evaluated its prognostic value in a training cohort. Immunohistochemical analysis of Cav-1 expression was scored as (++), (+) or (-) according to the proportion of positively stained tumor cells (T) and stromal cells (S). Correlation analysis between tumor/stromal Cav-1 expression and clinicopathological parameters revealed that only T(++) Cav-1 status was positively associated with tumor size and histological nodal status (P = 0.019 and 0.021, respectively). Univariate analysis revealed that combined T(++)/S(-) status was significantly correlated with unfavorable prognostic outcomes (P < 0.001). Multivariate analysis demonstrated that this combined status is an independent prognostic factor for primary breast cancer (P = 0.002). Clinical outcomes in different subgroups of breast cancer patients were also strictly dependent on this combined status (P < 0.05). The prognostic value of T(++)/S(-) Cav-1 status was also validated in the testing cohort. Collectively, our data indicate that high Cav-1 expression in tumor cells and lack of this expression in stromal cells could help identify a particular subgroup of breast cancer patients with potentially poor survival. Further studies are required to understand the regulatory mechanism of Cav-1 in the tumor microenvironment.     

Hedgehog overexpression is associated with stromal interactions and predicts for poor outcome in breast cancer

Hedgehog (Hh) signaling plays an important role in several malignancies but its clinical significance in breast cancer is unclear. In a cohort of 279 patients with invasive ductal carcinoma of the breast, expression of Hh ligand was significantly associated with increased risk of metastasis, breast cancer-specific death, and a basal-like phenotype. A paracrine signature, encompassing high epithelial Hh ligand and high stromal Gli1, was an independent predictor for overall survival in multivariate analysis. In 2 independent histological progression series (n = 301), Hh expression increased with atypia. Hh ligand overexpression in a mouse model of basal breast cancer increased growth, induced a poorly differentiated phenotype, accelerated metastasis, and reduced survival. A stromal requirement for these effects was supported by the lack of similar Hh-mediated changes in vitro, and by stromal-specific expression of Hh target genes in vivo. Furthermore, inhibition of Hh ligand with a monoclonal antibody (5E1) inhibited tumor growth and metastasis. These data suggest that epithelial-stromal Hh signaling, driven by ligand expression in carcinoma cells, promotes breast cancer growth and metastasis. Blockade of Hh signaling to peritumoral stromal cells may represent a novel therapeutic approach in some basal-like breast cancers.     

The humoral immune system has a key prognostic impact in node-negative breast cancer

Estrogen receptor (ER) expression and proliferative activity are established prognostic factors in breast cancer. In a search for additional prognostic motifs, we analyzed the gene expression patterns of 200 tumors of patients who were not treated by systemic therapy after surgery using a discovery approach. After performing hierarchical cluster analysis, we identified coregulated genes related to the biological process of proliferation, steroid hormone receptor expression, as well as B-cell and T-cell infiltration. We calculated metagenes as a surrogate for all genes contained within a particular cluster and visualized the relative expression in relation to time to metastasis with principal component analysis. Distinct patterns led to the hypothesis of a prognostic role of the immune system in tumors with high expression of proliferation-associated genes. In multivariate Cox regression analysis, the proliferation metagene showed a significant association with metastasis-free survival of the whole discovery cohort [hazard ratio (HR), 2.20; 95% confidence interval (95% CI), 1.40-3.46]. The B-cell metagene showed additional independent prognostic information in carcinomas with high proliferative activity (HR, 0.66; 95% CI, 0.46-0.97). A prognostic influence of the B-cell metagene was independently confirmed by multivariate analysis in a first validation cohort enriched for high-grade tumors (n = 286; HR, 0.78; 95% CI, 0.62-0.98) and a second validation cohort enriched for younger patients (n = 302; HR, 0.83; 95% CI, 0.7-0.97). Thus, we could show in three cohorts of untreated, node-negative breast cancer patients that the humoral immune system plays a pivotal role in metastasis-free survival of carcinomas of the breast.     

Glycan‐related gene expression signatures in breast cancer subtypes; relation to survival

Alterations in glycan structures are early signs of malignancy and have recently been proposed to be in part a driving force behind malignant transformation. Here, we explore whether differences in expression of genes related to the process of glycosylation exist between breast carcinoma subtypes – and look for their association to clinical parameters.Five expression datasets of 454 invasive breast carcinomas, 31 ductal carcinomas in situ (DCIS), and 79 non‐malignant breast tissue samples were analysed. Results were validated in 1960 breast carcinomas. 419 genes encoding glycosylation‐related proteins were selected.The DCIS samples appeared expression‐wise similar to carcinomas, showing altered gene expression related to glycosaminoglycans (GAGs) and N‐glycans when compared to non‐malignant samples. In‐situ lesions with different aggressiveness potentials demonstrated changes in glycosaminoglycan sulfation and adhesion proteins. Subtype‐specific expression patterns revealed down‐regulation of genes encoding glycan‐binding proteins in the luminal A and B subtypes. Clustering basal‐like samples using a consensus list of genes differentially expressed across discovery datasets produced two clusters with significantly differing prognosis in the validation dataset. Finally, our analyses suggest that glycolipids may play an important role in carcinogenesis of breast tumors – as demonstrated by association of B3GNT5 and UGCG genes to patient survival.In conclusion, most glycan‐specific changes occur early in the carcinogenic process. We have identified glycan‐related alterations specific to breast cancer subtypes including a prognostic signature for two basal‐like subgroups. Future research in this area may potentially lead to markers for better prognostication and treatment stratification of breast cancer patients.     

Overexpression of the oncogenic signal transducer Gab2 occurs early in breast cancer development

Gab2, a docking‐type signaling protein with demonstrated oncogenic potential, is overexpressed in breast cancer, but its prognostic significance and role in disease evolution remain unclear. Immunohistochemical detection of Gab2 in a large cohort of primary human breast cancers of known outcome revealed that while Gab2 expression was positively correlated with increased tumor grade, it did not correlate with disease recurrence or breast cancer‐related death in the total cohort or in patients stratified according to lymph node, estrogen receptor (ER) or HER2 status. Interestingly, analysis of a “progression series” that included premalignant and preinvasive breast lesions as well as samples of metastatic disease revealed that Gab2 expression was significantly enhanced in the earliest lesion examined, usual ductal hyperplasia, with a further increase detected in ductal carcinoma in situ (DCIS). Furthermore, expression was less in invasive cancers and lymph node metastases than in DCIS, but still higher than in normal breast. These findings indicate that while Gab2 expression is not prognostic in breast cancer, its role in early disease evolution warrants further analysis, as Gab2 and its effectors may provide targets for novel strategies aimed at preventing breast cancer development.