A prognostic gene expression profile that predicts circulating tumor cell presence in breast cancer patients

The detection of circulating tumor cells (CTCs) in the peripheral blood and microarray gene expression profiling of the primary tumor are two promising new technologies able to provide valuable prognostic data for patients with breast cancer. Meta-analyses of several established prognostic breast cancer gene expression profiles in large patient cohorts have demonstrated that despite sharing few genes, their delineation of patients into "good prognosis" or "poor prognosis" are frequently very highly correlated, and combining prognostic profiles does not increase prognostic power. In the current study, we aimed to develop a novel profile which provided independent prognostic data by building a signature predictive of CTC status rather than outcome. Microarray gene expression data from an initial training cohort of 72 breast cancer patients for which CTC status had been determined in a previous study using a multimarker QPCR-based assay was used to develop a CTC-predictive profile. The generated profile was validated in two independent datasets of 49 and 123 patients and confirmed to be both predictive of CTC status, and independently prognostic. Importantly, the "CTC profile" also provided prognostic information independent of the well-established and powerful '70-gene' prognostic breast cancer signature. This profile therefore has the potential to not only add prognostic information to currently-available microarray tests but in some circumstances even replace blood-based prognostic CTC tests at time of diagnosis for those patients already undergoing testing by multigene assays.     

Report of the National Oncology Research and Developement Consortium, 2003

Consorcial projects focused on 5 cancer types, breast-, colorectal-, head and neck- and pediatric cancers, and malignant melanoma. Breast cancer studies revealed unique splicing mechanisms concerning BRCA1. In sporadic breast cancers the involvement of DNA-repair genes was proved to be dependent on the histological type. Bone-metastatic tumors have been characterized by decreased NM23 and increased c-met and p53 expressions. C-erbB2 genotype of the primary tumor was not maintained frequently in bone metastases. Application of DNA-microarray and quantitative PCR technologies improved the prediction of therapeutic sensitivity of breast cancers. Colorectal cancer studies revealed regional inhomogenities (clusters) in various geographical regions of Hungary, which were distinct in the case of colonic and rectal cancers. To increase the sensitivity of fecal blood test of colorectal cancer screening, a new double-antibody test was developed and tested in a large cohort of patients. Genetic analysis revealed that hypermethylation is a significant factor in microsatellite instability which, and plays a role in silencing of APC and E-cadherin genes as well. The Hungarian pattern of TS polymorphism was also determined and was correlated not only with the efficacy of 5-FU treatment but with the progression of the disease as well. Population-based studies have been carried out in head and neck cancer patients (HNC) and smokers as well to reveal the genetic background of increasing tumor incidence. These studies revealed polymorphism in XRCC1/3 methylation enzyme gene which has preventive role. Other studies found frequent local immunosuppression in HNC patients. Studies indicated that the success of irradiation in this cancer type is dependent on the anti-vascular effects. Pediatric cancer studies determined the parameters of neuroblastoma screening based on VMA measurements. New splice variants of the WT1 gene involved in the monitoring of MRD of ALL patients was also described this year. We also obtained positive experimental data for the retinoic acid therapy of ALL. Melanoma studies extensively used DNA-microarray technology which identified 4 melanoma-specific and 2 melanoma progression-specific genes. In experimental human melanoma xenograft models we have identified 3 anti-metastatic agents: low molecular weight heparin, 2-methoxyestradiol and erythropoietin-alpha, where the later was characterized by specific effects on tumor vasculature.     

Rapid detection of CAA/CAG repeat polymorphism in the AIB1 gene using DHPLC

Denaturing high-performance liquid chromatography (DHPLC) has been used for rapid and accurate DNA mutation analysis; to extend the DNA fragment lengths analysis. Recently, polymorphism in polyglutamine-coding region of Amplified In Breast cancer gene 1 (AIB1) was analyzed as an independent genetic risk factor influencing breast cancer onset in carriers of mutation in breast cancer predisposing gene 1 (BRCA1). We have implemented efficient, cost-effective and rapid method for analysis of the AIB1 polyglutamine repeat polymorphism based on DHPLC analysis (WAVE system) of unlabeled PCR products. This strategy can be useful for genotyping of other trinucleotide repeat polymorphisms using DHPLC in medium/high throughput settings.     

Rapid detection of CAA/CAG repeat polymorphism in the AIB1 gene using DHPLC

Denaturing high-performance liquid chromatography (DHPLC) has been used for rapid and accurate DNA mutation analysis; to extend the DNA fragment lengths analysis. Recently, polymorphism in polyglutamine-coding region of Amplified In Breast cancer gene 1 (AIB1) was analyzed as an independent genetic risk factor influencing breast cancer onset in carriers of mutation in breast cancer predisposing gene 1 (BRCA1). We have implemented efficient, cost-effective and rapid method for analysis of the AIB1 polyglutamine repeat polymorphism based on DHPLC analysis (WAVE system) of unlabeled PCR products. This strategy can be useful for genotyping of other trinucleotide repeat polymorphisms using DHPLC in medium/high throughput settings.     

Improved breast cancer prognosis through the combination of clinical and genetic markers

MOTIVATION: Accurate prognosis of breast cancer can spare a significant number of breast cancer patients from receiving unnecessary adjuvant systemic treatment and its related expensive medical costs. Recent studies have demonstrated the potential value of gene expression signatures in assessing the risk of post-surgical disease recurrence. However, these studies all attempt to develop genetic marker-based prognostic systems to replace the existing clinical criteria, while ignoring the rich information contained in established clinical markers. Given the complexity of breast cancer prognosis, a more practical strategy would be to utilize both clinical and genetic marker information that may be complementary. METHODS: A computational study is performed on publicly available microarray data, which has spawned a 70-gene prognostic signature. The recently proposed I-RELIEF algorithm is used to identify a hybrid signature through the combination of both genetic and clinical markers. A rigorous experimental protocol is used to estimate the prognostic performance of the hybrid signature and other prognostic approaches. Survival data analyses is performed to compare different prognostic approaches. RESULTS: The hybrid signature performs significantly better than other methods, including the 70-gene signature, clinical makers alone and the St. Gallen consensus criterion. At the 90% sensitivity level, the hybrid signature achieves 67% specificity, as compared to 47% for the 70-gene signature and 48% for the clinical makers. The odds ratio of the hybrid signature for developing distant metastases within five years between the patients with a good prognosis signature and the patients with a bad prognosis is 21.0 (95% CI:6.5-68.3), far higher than either genetic or clinical markers alone. AVAILABILITY: The breast cancer dataset is available at www.nature.com and Matlab codes are available upon request.     

乳腺癌细胞EGFR表达水平与长春瑞滨敏感度的相关性研究

乳腺癌是较早开始个体化治疗的肿瘤之一。表皮细胞生长因子受体（epidermalgrowthfactorreceptor,EGFR）6~表达与乳腺癌组织学分期、生长速度呈正相关,可作为乳腺癌患者预后的指标之一。临床上,长春瑞滨（Vinorelbine,NVB）~要作为耐药性晚期乳腺癌的挽救性化疗药物,单药治疗亦具有一定疗效。该研究结果发现,乳腺癌组织EGFR表达与NVB的敏感性相关（P=0．001）,而与紫杉醇、阿霉素及5．氟尿嘧啶无相关性。EGFRFH性乳腺癌细胞MDA-MB-435s对NVB耐药,而EGFR阳性细胞MCF-7则敏感．但是EGFR中和性抗体会降低敏感性。进一步研究发现,NVB会引起MCF-7表面EGFI滚达上调,以及胞内ERK1／2激酶的磷酸化,且这一效应会被抗EGFR抗体部分抑制。研究结果表明,乳腺癌细胞对NVB的敏感性与膜表面EGFR表达水平相关,提示EGFR可作为NVB治疗敏感性的预测分子。     

Inference of patient‐specific subpathway activities reveals a functional signature associated with the prognosis of patients with breast cancer

Breast cancer is one of the most deadly forms of cancer in women worldwide. Better prediction of breast cancer prognosis is essential for more personalized treatment. In this study, we aimed to infer patient‐specific subpathway activities to reveal a functional signature associated with the prognosis of patients with breast cancer. We integrated pathway structure with gene expression data to construct patient‐specific subpathway activity profiles using a greedy search algorithm. A four‐subpathway prognostic signature was developed in the training set using a random forest supervised classification algorithm and a prognostic score model with the activity profiles. According to the signature, patients were classified into high‐risk and low‐risk groups with significantly different overall survival in the training set (median survival of 65 vs 106 months, P = 1.82e‐13) and test set (median survival of 75 vs 101 months, P = 4.17e‐5). Our signature was then applied to five independent breast cancer data sets and showed similar prognostic values, confirming the accuracy and robustness of the subpathway signature. Stratified analysis suggested that the four‐subpathway signature had prognostic value within subtypes of breast cancer. Our results suggest that the four‐subpathway signature may be a useful biomarker for breast cancer prognosis.     

Financial aspects of targeted therapy of lung cancer as compared to conventional chemotherapy

Lung cancer is the leading cause of cancer death worldwide, and it is responsible for 20-25% of all cancer deaths. In Hungary, more than 4,000 lung cancer patients receive chemotherapy every year, and of them 3,000 suffer from non-small cell lung cancer. Despite the rapid development in antitumor treatment options, the response rates of chemotherapies in non-small cell lung cancer still remain between 15-35%. Recently, EGFR tyrosine kinase inhibitor therapy has been introduced with a response rate of 15% when used in unselected patients. However, this ratio may increase up to 70% when only patients with tumors containing EGFR mutation are treated. Therefore, results of translational research that could help pulmonologists and oncologists to apply tailored therapy in lung cancer patients are of great importance. The purpose of our work was to establish a model in order to study the relevance of immunohistochemical and molecular biological analyses of tumor specimens in treatment and patient selection, and to investigate their cost-sparing effects. We concluded that the most cost-effective type of patient selection could be achieved with EGFR mutation status analysis of the tumor tissue. Our results may help to determine the most cost-effective way of patient selection in case of non-small cell lung cancer patients requiring second line therapy; moreover, they might serve as a basis for further economic analyses.     

Report on the first year of the activity of the National Oncological RD Consortium

The project focuses on cancer types with outstanding publich health importance (breast-, colorectalhead and neck cancers and childhood tumors). Epidemiological studies revealed significant regional differences in the mobidity/mortality of these cancer types in Hungary. Molecular epidemiological studies revealed characteristic BRCA1 mutation patterns of familiar breast cancer and DNA repair enzyme polymorphism in head and neck cancer. New methods have been developed for the screening (lactoferrin), prognostication (c-met expression) or the prediction of therapeutic sensitivity (TS expression) of colorectal cancer. In the pediatric oncology program alternative way of MRD monitoring (WT1 expression) and a potential new therapeutic modality (IFN-alpha) of ALL was developed. Experimental studies demonstrated that the tumoral matrix significantly influences the effects of the classic chemotherapeutic agents. We have identified several genes the expression of which could serve efficiently as markers or targets for therapy of the progression of melanoma (alphaIIbbeta3 integrin, CD44v3 and decorin proteoglycans, AMF receptor).     

Prognostic significance of autophagy related genes in estrogen receptor positive tamoxifen treated breast cancer

Resistance to Tamoxifen constitutes a major therapeutic challenge in treating hormone sensitive breast cancer. The induction of autophagy has been shown to be involved as one of the mechanism responsible for Tamoxifen resistance. Autophagy related gene (ATG) members are the regulators and effectors of Macroautophagy process in the cellular systems. In this study, we evaluated the prognostic significance of ATGs in Tamoxifen treated breast cancer. The "Kaplan- Meier plotter" database was utilized to analyze the relevance and significance of ATGs mRNA expression to Relapse Free Survival in breast cancer patients. We used the data of patients who are Estrogen receptor positive and are treated with Tamoxifen. Hazard ratio and log-rank p-value were calculated using KM survival plots for various ATGs. Overexpressed ATG3, ATG 5, ATG 8B and PIK3R4 resulted in a poor prognosis. A gene signature of these ATGs predicts deteriorated RFS (p-value=8.3e-05 and HR=1.84 (1.35-2.51) and Distant Metastasis Free Survival (p value = 0.0027 and HR=2.03 (1.27-3.26). We report the distinct prognostic values of ATGs in patients of breast cancer treated with Tamoxifen. Thus, better understandings of the induction of autophagy pathway may potentially form the basis for use of autophagy inhibitors in the Tamoxifen treated breast cancer.     

A 17-marker panel for global genomic instability in breast cancer

Genomic instability is a hallmark of cancer that plays a pivotal role in breast cancer development and evolution. A number of existing prognostic gene expression signatures for breast cancer are based on proliferation-related genes. Here, we identified a 17-marker panel associated with genome stability. A total of 136 primary breast carcinomas were stratified by genome stability. Matched gene expression profiles showed an innate segregation based on genome stability. We identified a 17-marker panel stratifying the training and validation cohorts into high- and low-risk patients. The 17 genes associated with genomic instability strongly impacted clinical outcome in breast cancer. Pathway analyses determined chromosome organisation, cell cycle regulation, and RNA processing as the underlying biological processes, thereby offering options for drug development and treatment tailoring. Our work supports the applicability of the 17-marker panel to improve clinical outcome prediction for breast cancer patients based on a signature accounting for genomic instability.     

An estrogen receptor (ER)‐related signature in predicting prognosis of ER‐positive breast cancer following endocrine treatment

Quite a few estrogen receptor (ER)‐positive breast cancer patients receiving endocrine therapy are at risk of disease recurrence and death. ER‐related genes are involved in the progression and chemoresistance of breast cancer. In this study, we identified an ER‐related gene signature that can predict the prognosis of ER‐positive breast cancer patient receiving endocrine therapy. We collected RNA expression profiling from Gene Expression Omnibus database. An ER‐related signature was developed to separate patients into high‐risk and low‐risk groups. Patients in the low‐risk group had significantly better survival than those in the high‐risk group. ROC analysis indicated that this signature exhibited good diagnostic efficiency for the 1‐, 3‐ and 5‐year disease‐relapse events. Moreover, multivariate Cox regression analysis demonstrated that the ER‐related signature was an independent risk factor when adjusting for several clinical signatures. The prognostic value of this signature was validated in the validation sets. In addition, a nomogram was built and the calibration plots analysis indicated the good performance of this nomogram. In conclusion, combining with ER status, our results demonstrated that the ER‐related prognostic signature is a promising method for predicting the prognosis of ER‐positive breast cancer patients receiving endocrine therapy.     

RB-pathway disruption in breast cancer

In breast cancer, inactivation of the RB tumor suppressor gene is believed to occur via multiple mechanisms to facilitate tumorigenesis. However, the prognostic and predictive value of RB status in disease-specific clinical outcomes has remained uncertain. We investigated RB pathway deregulation in the context of both ER-positive and ER-negative disease using combined microarray datasets encompassing over 900 breast cancer patient samples. Disease-specific characteristics of RB pathway deregulation were investigated in this dataset by evaluating correlation among pathway genes as well as differential expression across patient tumor populations defined by ER status. Survival analysis among these breast cancer samples demonstrates that the RB-loss signature is associated with poor disease outcome within several independent cohorts. Within the ER-negative subpopulation, the RB-loss signature is associated with improved response to chemotherapy and longer relapse-free survival. Additionally, while individual genes in the RB target signature closely reproduce its prognostic value, they also serve to predict and monitor response to therapeutic compounds, such as the cytostatic agent PD-0332991. These results indicate that the RB-loss signature expression is associated with poor outcome in breast cancer, but predicts improved response to chemotherapy based on data in ER-negative populations. While the RB-loss signature, as a whole, demonstrates prognostic and predictive utility, a small subset of markers could be sufficient to stratify patients based on RB function and inform the selection of appropriate therapeutic regimens.     

A prognostic eight-lncRNA expression signature in predicting recurrence of ER-positive breast cancer receiving endocrine therapy

Long noncoding RNAs (lncRNAs) have the main role in the tumorigenesis of breast cancer. In the present study, lncRNA expression profiling was collected to identify a lncRNA expression signature from the Gene Expression Omnibus database. An eight-lncRNA signature was established to predict the survival of patients with estrogen receptor (ER)-positive breast cancer receiving endocrine therapy. Patients were separated into a low-risk group and a high-risk group based on this signature. Patients in high-risk group have worse survival compared to those in low-risk group using Kaplan–Meier curve analysis with log-rank test. Receiver operating characteristic analysis suggested good diagnostic efficiency of the eight-lncRNA signature. When adjusting the clinical features, including age, grade, lymph node status, and tumor size, this signature was independently associated with the relapse-free survival. The prognostic value of the lncRNA prognostic model was then validated in validation sets. When validated in a cohort of patients treated with neoadjuvant chemotherapy and endocrine therapy, this signature demonstrated good performance as well. Besides, we have built a nomogram that integrated the conventional clinicopathological features and the eight-lncRNA-based signature. To sum up, our results indicated that the eight-lncRNA prognostic model was a reliable tool to group patients at high and low risk of disease relapse. This signature may have possible implication in prognostic evaluations of patients with ER-positive breast cancer receiving endocrine therapy.     

RB-pathway disruption in breast cancer: Differential association with disease subtypes,disease-specific prognosis and therapeutic response

In breast cancer, inactivation of the RB tumor suppressor gene is believed to occur via multiple mechanisms to facilitate tumorigenesis. However, the prognostic and predictive value of RB status in disease-specific clinical outcomes has remained uncertain. We investigated RB pathway deregulation in the context of both ER-positive and ER-negative disease using combined microarray datasets encompassing over 900 breast cancer patient samples. Disease-specific characteristics of RB pathway deregulation were investigated in this dataset by evaluating correlation among pathway genes as well as differential expression across patient tumor populations defined by ER status. Survival analysis among these breast cancer samples demonstrates that the RB-loss signature is associated with poor disease outcome within several independent cohorts. Within the ER-negative subpopulation, the RB-loss signature is associated with improved response to chemotherapy and longer relapse-free survival. Additionally, while individual genes in the RB target signature closely reproduce its prognostic value, they also serve to predict and monitor response to therapeutic compounds, such as the cytostatic agent PD-0332991. These results indicate that the RB-loss signature expression is associated with poor outcome in breast cancer, but predicts improved response to chemotherapy based on data in ER-negative populations. While the RB-loss signature, as a whole, demonstrates prognostic and predictive utility, a small subset of markers could be sufficient to stratify patients based on RB function and inform the selection of appropriate therapeutic regimens.Key words: RB, breast cancer, microarray, proliferation, cytostatics     

Plasminogen activator inhibitor-1 (PAI-1) gene 4G/5G promoter polymorphism is not associated with breast cancer

The antigen content of plasminogen activator inhibitor-1 (PAI-1) in primary breast cancer tissue extracts may be of strong prognostic value: high levels of PAI-1 in tumors predict poor prognosis for patients. The gene encoding PAI-1 is highly polymorphic and an insertion (5G)/deletion (4G) polymorphism in the PAI-1 gene promoter (the 4G/5G polymorphism), may have functional significance in PAI-1 expression. In the present work the distribution of genotypes and frequency of alleles of the 4G/5G polymorphism in subjects with breast cancer were investigated. Tumor tissues were obtained from 100 postmenopausal women with node-negative and node-positive ductal breast carcinoma with uniform tumor size. Blood samples from age matched healthy women served as control. The 4G/5G polymorphism was determined by PCR amplification using the allele specific primers. The distribution of the genotypes of the 4G/5G polymorphism in both control and patients did not differ significantly (P > 0.05) from those predicted by the Hardy-Weinberg distribution. There were no differences in the genotype distributions and allele frequencies between node-positive and node-negative patients. The 4G/5G polymorphism may not be linked with elevated level of PAI-1 observed in breast cancer and therefore may not be associated with appearance and/or progression of breast cancer.     

Significant alterations of the novel 15 gene signature identified from macrophage-tumor interactions in breast cancer

Background

Tumor microenvironment is composed of a largely altered extracellular matrix with different cell types. The complex interplay between macrophages and tumor cells through several soluble factors and signaling is an important factor in breast cancer progression.