Comprehensive molecular biomarker identification in breast cancer brain metastases

Background

Breast cancer brain metastases (BCBM) develop in about 20–30% of breast cancer (BC) patients. BCBM are associated with dismal prognosis not at least due to lack of valuable molecular therapeutic targets. The aim of the study was to identify new molecular biomarkers and targets in BCBM by using complementary state-of-the-art techniques.

Methods

We compared array expression profiles of three BCBM with 16 non-brain metastatic BC and 16 primary brain tumors (prBT) using a false discovery rate (FDR) p < 0.05 and fold change (FC) > 2. Biofunctional analysis was conducted on the differentially expressed probe sets. High-density arrays were employed to detect copy number variations (CNVs) and whole exome sequencing (WES) with paired-end reads of 150 bp was utilized to detect gene mutations in the three BCBM.

Results

The top 370 probe sets that were differentially expressed between BCBM and both BC and prBT were in the majority comparably overexpressed in BCBM and included, e.g. the coding genes BCL3, BNIP3, BNIP3P1, BRIP1, CASP14, CDC25A, DMBT1, IDH2, E2F1, MYCN, RAD51, RAD54L, and VDR. A number of small nucleolar RNAs (snoRNAs) were comparably overexpressed in BCBM and included SNORA1, SNORA2A, SNORA9, SNORA10, SNORA22, SNORA24, SNORA30, SNORA37, SNORA38, SNORA52, SNORA71A, SNORA71B, SNORA71C, SNORD13P2, SNORD15A, SNORD34, SNORD35A, SNORD41, SNORD53, and SCARNA22. The top canonical pathway was entitled, role of BRCA1 in DNA damage response. Network analysis revealed key nodes as Akt, ERK1/2, NFkB, and Ras in a predicted activation stage. Downregulated genes in a data set that was shared between BCBM and prBT comprised, e.g. BC cell line invasion markers JUN, MMP3, TFF1, and HAS2. Important cancer genes affected by CNVs included TP53, BRCA1, BRCA2, ERBB2, IDH1, and IDH2. WES detected numerous mutations, some of which affecting BC associated genes as CDH1, HEPACAM, and LOXHD1.

Conclusions

Using complementary molecular genetic techniques, this study identified shared and unshared molecular events in three highly aberrant BCBM emphasizing the challenge to detect new molecular biomarkers and targets with translational implications. Among new findings with the capacity to gain clinical relevance is the detection of overexpressed snoRNAs known to regulate some critical cellular functions as ribosome biogenesis.

     

Her2 genotype and breast cancer progression in Korean women

The amplification and overexpression of Her2 proto-oncogene have been found to be associated with the development and progression of human breast cancer. A polymorphic valine allele at codon 655 of the Her2 gene (Her2(V655)) was suggested by some authors to be a susceptible genetic factor for the development of breast cancer. The Her2 polymorphism at codon 655 was investigated in 304 Korean women including 177 patients with breast cancer. The association between Her2 genotype and Her2 protein overexpression was also examined in breast cancers by immunohistochemistry. Her2(V655) was not associated with a significant breast cancer risk (odds ratio (OR), 1.792; 95% confidence interval (CI), 0.459-6.991). The frequency of homozygous or heterozygous valine allele increased in stage 2 patients (OR, 1.67; 95% CI, 0.67-4.19), and patients in stages 3 and 4 (OR, 3.36; 95% CI, 0.85-13.42) compared to patients in stage 0. However, an association between the presence of the valine allele and the overexpression of Her2 protein could not be demonstrated. These results suggest that Her2 polymorphism at codon 655 is not associated with the development of breast cancer in Korean women. However, there is a possibility that the valine allele at codon 655 might be related to increased risk of breast cancer progression.     

Identification of the key pathways and genes involved in HER2-positive breast cancer with brain metastasis

BackgroundThe risk of brain metastasis (BM) in HER2-positive (+) breast cancer (BC) patients is significantly higher than that in HER2-negative (-) BC patients. The high incidence and mortality rate makes it urgent to elucidate the key pathways and genes involved and identify patients who are more at risk of developing BM.Materials and methodsTo identify the target genes in HER2+BC patients with BM, we analyzed the microarray datasets (GSE43837) derived from the Gene Expression Omnibus (GEO) database. The GEO2R tool was used to extract the differentially expressed genes (DEGs) involved in HER2+ primary BC and BC with BM. Bioinformatics methods including Gene Ontology (GO) functional annotation analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed with the screened DEGs. The protein-protein interactions of the DEGs were analyzed using the Search Tool for the Retrieval of Interacting Genes (STRING) database and visualized using Cytoscape software. Finally, GSEA analysis was performed to identify the hub genes and the important pathways.ResultsA total of 751 upregulated and 285 downregulated DEGs were identified. The GO function and KEGG pathway enrichment analyses indicated that the DEGs were all enriched in the protein binding molecular function. The top five hub nodes were screened out, included PHLPP1, UBC, ACACB, TGFB1, and ACTB. The GSEA results demonstrated that the five hub genes are mainly enriched in the ribosomal pathway.ConclusionOur study suggests that the five hub genes (PHLPP1, UBC, ACACB, TGFB1, and ACTB) are associated with HER2+BC with BM. The GSEA analysis revealed that the ribosomal pathway seems to play a very important role in the pathogenesis of HER2+BC with BM.     

Her2在乳腺癌和胃癌中表达的临床意义

目的：探讨Her2在乳腺癌和胃癌中表达的临床情况与相关意义,为肿瘤防治提供参考。方法：收集2009年3月到2014年5月在我院行外科手术切除的60例确诊的进展期胃癌标本与60例确诊的晚期乳腺癌标本,都进行Her2病理表达免疫组化分析,并调查了相关临床病理资料进行相关性分析。结果：Her2的乳腺癌和胃癌标本中阳性表达率分别为400%和367%,对比差异无统计学意义(P>005)。Her2表达与恶性肿瘤患者的年龄、病理类型无关,与TNM分期与淋巴结转移有明显相关性(P<005)。Spearman相关分析显示乳腺癌与胃癌中的Her2表达都与Survivin、Bcl 2表达存在明显正向相关性(P<005)。结论：乳腺癌和胃癌患者Her2都呈现高病理表达状况,与TNM分期与淋巴结转移有明显相关性,能通过调节部分凋亡抑制蛋白,参与肿瘤的发病与转移过程。     

Targeted delivery of PLK1-siRNA by ScFv suppresses Her2+ breast cancer growth and metastasis

A major obstacle to developing small interfering RNAs (siRNAs) as cancer drugs is their intracellular delivery to disseminated cancer cells. Fusion proteins of single-chain fragmented antibodies (ScFvs) and positively charged peptides deliver siRNAs into specific target cells. However, the therapeutic potential of ScFv-mediated siRNA delivery has not been evaluated in cancer. Here, we tested whether Polo-like kinase 1 (PLK1) siRNAs complexed with a Her2-ScFv-protamine peptide fusion protein (F5-P) could suppress Her2(+) breast cancer cell lines and primary human cancers in orthotopic breast cancer models. PLK1-siRNAs transferred by F5-P inhibited target gene expression, reduced proliferation, and induced apoptosis of Her2(+) breast cancer cell lines and primary human cancer cells in vitro without triggering an interferon response. Intravenously injected F5-P/PLK1-siRNA complexes concentrated in orthotopic Her2(+) breast cancer xenografts and persisted for at least 72 hours, leading to suppressed PLK1 gene expression and tumor cell apoptosis. The intravenously injected siRNA complexes retarded Her2(+) breast tumor growth, reduced metastasis, and prolonged survival without evident toxicity. F5-P-mediated delivery of a cocktail of PLK1, CCND1, and AKT siRNAs was more effective than an equivalent dose of PLK1-siRNAs alone. These data suggest that F5-P could be used to deliver siRNAs to treat Her2(+) breast cancer.     

Metastatic profiling of HER2-positive breast cancer cell lines in xenograft models

Most studies on breast cancer metastasis have been performed using triple-negative breast cancer cells; thus, subtype-dependent metastatic ability of breast cancer is poorly understood. In this research, we performed intravenous injection (IVI) and intra-caudal arterial injections using nine human epidermal growth factor receptor-2 (HER2)-positive breast cancer cell lines for evaluating their metastatic abilities. Our results showed that MDA-MB-453, UACC-893, and HCC-202 had strong bone metastatic abilities, whereas HCC-2218 and HCC-1419 did not show bone metastasis. HER2-positive cell lines could hardly metastasize to the lung through IVI. From the genomic analysis, gene signatures were extracted according to the breast cancer subtypes and their metastatic preferences. The UACC-893 cell line was identified as a useful model for the metastasis study of HER2-positive breast cancer. Combined with our previous result on brain metastasis ability, we provide a characteristic metastasis profile of HER2-positive breast cancer cell lines in this study.

     

Risk factors and survival outcomes in patients with brain metastases from breast cancer

Development of central nervous system (CNS) metastases in breast cancer (BC) is associated with poor prognosis. The incidence of CNS metastases in metastatic BC is reported to be about 10–16 %, but different subtypes of BC are associated with different risk of developing CNS metastases. We retrospectively analysed the risk of CNS metastases and the outcome in a cohort of 473 patients with metastatic BC. CNS metastases were diagnosed in 15.6 % of patients and median survival from diagnosis of CNS metastases was 7.53 (25th–75th 2.8–18.9) months. The risk of developing CNS metastases was higher in patients with grade 3, hormone receptor negative, HER2-positive, high Ki-67 BC. When compared to luminal A subtype, only HER2-positive BC was associated with increased risk of CNS metastases. Survival from diagnosis of CNS metastases was longer in patients with HER2-positive BC, while it was shorter in patients that did not receive any locoregional treatment, or with extra-CNS disease, or with more than 3 CNS lesions.     

ESR1-promoter-methylation status in primary breast cancer and its corresponding metastases

The role of ESR1 methylation in breast cancer and its influence on disease progression is not yet fully understood. Healthy breast tissue usually does not show ESR1 promoter methylation, whereas the frequency of ESR1 methylation appears to increase in primary breast cancer and in metastatic disease. Although women with ER positive breast cancer have a good prognosis, some will relapse. We aimed to evaluate the methylation status of ESR1 in primary breast cancer and its corresponding metastases by a methylation-specific real-time PCR and to correlate the methylation status with clinical outcome. Women who were treated for primary and metastatic breast cancer were included in the study. Tumor DNA was isolated from paraffin embedded tissue sections. After bisulfite treatment ESR1 promoter methylation was analyzed by real time-MSP of each tissue sample. Kaplan–Meier–Curves were drawn for survival. In the group of patients with positive ESR1 promoter methylation in the primary breast carcinoma survival was lower compared to the group of patients without methylation (38.1 months vs. 54.3 months, n.s.). Seven out of 19 (37%) of those patients with positive ESR1 promoter methylation developed loss of ER expression in metastatic disease. None of the patients who had primary tumours that were ESR1 methylation negative developed ER expression negative metastatic disease. The results underline the importance of the ESR1 promoter methylation and its potential application as a predictive marker. To improve the clinical outcome of patients with metastatic disease, those with initially positive ESR1 methylation status should undergo a tissue biopsy already at the beginning of metastatic disease to identify those with loss of ER expression and thus resitance to anti-endocrine therapy.     

Long term disease-free survival and T cell and antibody responses in women with high-risk Her2+ breast cancer following vaccination against Her2

Background  

The HER2-inhibiting antibody trastuzumab, in combination with chemotherapy, significantly improves survival of women with resected, HER2-overexpressing breast cancers, but is associated with toxicities including a risk of cardiomyopathy. Additionally, the beneficial effect of trastuzumab is expected to decrease once the drug is discontinued. We proposed to address these concerns by using cancer vaccines to stimulate HER2 intracellular domain (ICD)-specific T cell and antibody responses.     

Vascular endothelial growth factor expression promotes the growth of breast cancer brain metastases in nude mice

Patients with breast cancer brain metastases cannot be cured and have a poor prognosis, with a median survival time of six months after diagnosis, despite developments in diagnostic and therapeutic modalities. In large part the progress in understanding the biology of breast cancer brain metastasis has been limited by the lack of suitable cell lines and experimental models. The objective of this study was to develop a reliable experimental model to study the pathogenesis of breast cancer brain metastases, using intra-internal carotid artery injection of breast cancer cells into nude mice. Brain metastasis-selected variant cells were recovered after three cycles of injection into the internal carotid artery of nude mice and harvest of brain metastases, resulting in variants termed MDA-231 BR1, -BR2 and -BR3. The metastasis-selected cells had increased potential for experimental brain metastasis and mice injected with these cells had significantly shorter mean survival than mice injected with the original cell line. Brain metastatic lesions of the selected variants contained significantly more CD31-positive blood vessels than metastases of the non-selected cell line. The variants selected from brain metastases released significantly more VEGF-A and IL-8 into culture supernatants than the original cell line, and more VEGF-A RNA when cultured in normoxic conditions. Mice injected with MDA-231 BR3 into the carotid artery were treated with the VEGF-receptor tyrosine kinase inhibitor PTK787/Z 222584. Oral administration of the inhibitor resulted in a significant decrease in brain tumor burden, reduced CD31-positive vessels in the brain lesions and incidence of PCNA positive tumor cells, and increased apoptosis in the tumor, as measured by TUNEL labeling. We conclude that elevated VEGF expression contributes to the ability of breast cancer cells to form brain metastases. Targeting endothelial cells with a VEGF-receptor specific tyrosine kinase inhibitor reduced angiogenesis and restricted the growth of the brain metastases.     

Expression and amplification of Her2, EGFR and cyclin D1 in breast cancer: immunohistochemistry and chromogenic in situ hybridization

Determination of Her2, epidermal growth factor receptor (EGFR) and cyclin D1 status is now of major clinical importance due to the development of molecule-targeting drugs in anticancer therapy. Immunohistochemistry (IHC) and chromogenic in situ hybridization (CISH) are the most simple and convenient methods for evaluating gene alterations and their protein consequences. The purpose of the present study was to investigate the status of Her2, EGFR and cyclin D1 on both IHC and CISH in 95 primary breast carcinomas. There was substantial consistency between the IHC and CISH results of Her2 and EGFR, showing fair agreement between protein overexpression and gene amplification. However, cyclin D amplification was not related to protein overexpression. Moreover, there was no correlation between Her2, EGFR and cyclin D1. Her2 protein overexpression and amplification were positively associated with histological grade, nuclear grade and inversely correlated with the expression of estrogen receptor (ER) and progesterone receptor (PR). In ER-negative and postmenopausal patients, EGFR gene amplification was strongly associated with worse recurrence-free survival (P = 0.0087, P = 0.0149, respectively). Overall, the present findings suggest that EGFR gene amplification is important in predicting prognosis and this should be evaluated in breast carcinoma in addition to Her2 status in routine pathological practice.     

Prognostic significance of cytokeratin-positive breast cancer metastases

The most important discriminant in staging carcinoma of the breast is the presence of positive axillary lymph nodes. In this study, we determined if 45 female breast cancer patients originally classified as lymph node-negative by standard light microscopy (SLM) could be more accurately classified by immunohistochemical (IH) examination of their lymph nodes with an anticytokeratin monoclonal antibody cocktail. Identical sections of lymph nodes were sequentially examined by SLM and IH. Eight nodes (1%) in a total of five patients (11%) were positive by SLM. In comparison, 12 nodes (1.5%) in a total of nine patients (20%) were positive by IH. Five nodes were positive by IH and negative by SLM. There was no correlation between IH-detected metastases and tumor size or patient age. The survival curve for patients with IH-detected metastases was significantly worse than that of patients without IH-detected metastases. IH detection methods may be an important adjunct in staging breast cancer patients.