Optimized high-throughput microRNA expression profiling provides novel biomarker assessment of clinical prostate and breast cancer biopsies

Background  

Recent studies indicate that microRNAs (miRNAs) are mechanistically involved in the development of various human malignancies, suggesting that they represent a promising new class of cancer biomarkers. However, previously reported methods for measuring miRNA expression consume large amounts of tissue, prohibiting high-throughput miRNA profiling from typically small clinical samples such as excision or core needle biopsies of breast or prostate cancer. Here we describe a novel combination of linear amplification and labeling of miRNA for highly sensitive expression microarray profiling requiring only picogram quantities of purified microRNA.     

Comparison of cryopreservation and standard needle biopsy for gene expression profiling of human breast cancer specimens

Summary Given the growing importance of molecular profiling of breast cancer, we initiated a small study to test whether human breast biopsies obtained via cryopreservation large core needle biopsy (C-LCNB) provided similar gene expression profiles compared with optimally handled standard large core needle biopsy (S-LCNB) specimens. Five matched pairs of C-LCNB versus S-LCNB were obtained at the same visit, and subjected to gene array expression analysis using the Affymetrix system with U133A chips. No significant changes in gene expression were identified comparing the C-LCNB versus the matched S-LCNB from individual patients. This was corroborated by a paired t-test analysis, which supported the hypothesis that the S/C biopsies measured equivalent samples. A small number of genes (17) showed decreased expression when second biopsies were compared with first biopsies, suggesting a slight patient response to the first biopsy. A scatter plot analysis comparing first biopsy versus second biopsy values disclosed a slope of 0.859, further indicating that the first biopsy affects the second biopsy measurement. It therefore appears that conventional biopsies, when handled appropriately, provide RNA which is equivalent to RNA from biopsies which are frozen immediately, but that multiple biopsy protocols may introduce additional complexities.     

Radiation-induced effects on gene expression: an in vivo study on breast cancer.

BACKGROUND AND PURPOSE: Breast cancer is diagnosed worldwide in approximately one million women annually and radiation therapy is an integral part of treatment. The purpose of this study was to investigate the molecular basis underlying response to radiotherapy in breast cancer tissue. MATERIAL AND METHODS: Tumour biopsies were sampled before radiation and after 10 treatments (of 2 Gray (Gy) each) from 19 patients with breast cancer receiving radiation therapy. Gene expression microarray analyses were performed to identify in vivo radiation-responsive genes in tumours from patients diagnosed with breast cancer. The mutation status of the TP53 gene was determined by using direct sequencing. RESULTS AND CONCLUSION: Several genes involved in cell cycle regulation and DNA repair were found to be significantly induced by radiation treatment. Mutations were found in the TP53 gene in 39% of the tumours and the gene expression profiles observed seemed to be influenced by the TP53 mutation status.     

Direct comparison of microarray gene expression profiles between non-amplification and a modified cDNA amplification procedure applicable for needle biopsy tissues

Global gene expression profiling by cDNA microarray analysis has been used to discover the biomarkers for early diagnosis of various cancers, subclassing cancer type, and prediction of patient's treatment outcome. The information provided by gene expression profiling may contribute to the design of molecular mechanism-based strategies for cancer prevention and/or treatment. However, the standard procedure for cDNA microarray analysis requires 5mug of good quality total RNA as starting material for each target preparation reaction. Thus, there is a limit for needle biopsy samples, laser capture microdissected tissues, or flow-sorted cells to successfully utilize the microarray technology. In order to profile the gene expression of needle biopsy tissue, we have modified the standard protocol by carrying out cDNA amplification after cDNA synthesis. We compared percentage present calls, absent calls, reproducibility, and concordance in needle biopsy samples processed by standard microarray protocol (cDNA non-amplification method) and our modified protocol (cDNA amplification method). The results showed that cDNA amplification method provided high reproducibility, representation, and concordance with the standard cDNA non-amplification method. We have successfully analyzed the gene expression profiles of needle biopsy tissues using the modified method without significantly changing the expression profiles. These results suggest that the global gene expression profiles of small biopsy samples can be achieved by our modified method to facilitate the analysis of gene expression profiles for clinical application.     

Gene expression profiling of NMU-induced rat mammary tumors: cross species comparison with human breast cancer

Breast cancer is a complex genetic disease characterized by the accumulation of multiple molecular alterations. The NMU breast cancer model induced in the rat is used for the study of mammary carcinogenesis because it closely mimics human breast disease. To assess the validity of this model from a more global molecular perspective, and also to devise a general technique to compare animal profiles with human microarray studies, we have characterized 25 NMU-induced mammary tumors and 11 normal glands using a combination of immunohistochemical and microarray analyses. The rat mammary carcinomas were classified as non-invasive, ER-positive ductal carcinomas with a composition of differentiated epithelial and myoepithelial cell lineages. Gene expression profiles generated using rat Affymetrix arrays containing 15,866 genes demonstrated that the rat mammary tumors are homogeneous and that H-ras mutations did not confer a unique molecular signature. We compared the resulting rat profiles with those obtained from a human dataset by merging the raw microarray data, using an approach that involves a combination of cross-species and cross-platform analysis. Using this novel strategy, we demonstrate the ability of 2305 rat orthologs to recapitulate the classification of human tumors derived from human Affymetrix arrays. The gene expression profiles of the NMU-induced primary tumors were most similar to ER-positive, low to intermediate grade breast cancer. Our technique provides a means to correlate gene expression data from animal models of cancer to human cancer and disease states.     

Protein Tyrosine Phosphatase 4A2 Expression Predicts Overall and Disease-Free Survival of Human Breast Cancer and Is Associated with Estrogen and Progestin Receptor Status

Expression of protein tyrosine phosphatase PTP4A2 (also known as PRL2) has been examined in a variety of human carcinomas, although its role in breast cancer remains inconclusive. Since the majority of previous breast cancer studies utilized tissue biopsies composed of heterogeneous cell populations, we hypothesized that an examination of PTP4A2 expression in carcinoma cells isolated by laser capture microdissection (LCM) would provide a more accurate means of assessing its predictive value. From investigations of 247 human breast cancer biopsies collected under standardized, stringent conditions, total RNA was extracted from LCM-procured carcinoma cells to perform microarray analyses to identify gene signatures associated with breast cancer behavior. Expression of PTP4A2 was corroborated by real-time quantitative polymerase chain reaction (qPCR) and referenced to estrogen and progesterone receptor levels. Patient outcomes for overall and disease-free survival were more favorable (p?=?0.004 and p?=?0.001, respectively) when the expression of PTP4A2 in breast carcinomas was increased compared to patients with biopsies with decreased PTP4A2 levels. PTP4A2 expression determined either by microarray or qPCR was elevated in either estrogen receptor (ER)-positive or progestin receptor (PR)-positive breast cancer biopsies compared to ER-negative or PR-negative biopsies. However, PTP4A2 expression was only correlated with overall survival in PR-positive breast carcinomas. These data suggest that PTP4A2 mRNA expression alone may serve as a biomarker for prediction of a breast cancer patient’s risk of recurrence and overall survival.     

Gene expression profiles derived from fine needle aspiration correlate with response to systemic chemotherapy in breast cancer

Background  

Drug resistance in breast cancer is a major obstacle to successful chemotherapy. In this study we used cDNA microarray technology to examine gene expression profiles obtained from fine needle aspiration (FNA) of primary breast tumors before and after systemic chemotherapy. Our goal was to determine the feasibility of obtaining representative expression array profiles from limited amounts of tissue and to identify those expression profiles that correlate with treatment response.     

Application of oligonucleotide microarrays to assess the biological effects of neoadjuvant imatinib mesylate treatment for localized prostate cancer.

PURPOSE: Neoadjuvant administration of antineoplastic therapies is used to rapidly assess the clinical and biological activity of novel systemic treatments. To assess the feasibility of using microarrays to assess molecular end points following targeted treatment in a heterogeneous tumor, we measured global gene expression in localized prostate cancer before and following neoadjuvant treatment with imatinib mesylate. PATIENTS AND METHODS: Patients with intermediate-risk to high-risk prostate cancer were treated for 6 weeks with 200 to 300 mg of oral imatinib mesylate. Frozen tissue was obtained from pretreatment ultrasound-guided biopsies and posttreatment radical prostatectomy specimens. Oligonucleotide microarray analysis following laser capture microdissection (LCM) and RNA amplification was used to assess gene expression changes associated with imatinib mesylate therapy. Immunohistochemistry was used to measure protein expression of MKP1 and CD31 and to assess cellular apoptosis. RESULTS: Of the 11 patients enrolled, high-quality microarray data was obtained from both biopsies (n = 7) and radical prostatectomy specimens (n = 9). Technically introduced intrasample gene expression variability was found to be significantly less than intertumor biological variability. Large gene expression differences were observed, and the gene with the most consistent differential expression (MKP1) was validated by immunohistochemistry. Gene set enrichment analysis suggests that imatinib mesylate therapy results in apoptosis of microvascular endothelial cells, an observation anecdotally supported by immunohistochemistry. CONCLUSIONS: This study shows that high-quality microarray data can be generated using LCM and RNA amplification to discover potential mechanisms of targeted therapy in cancer.     

Validation of tissue microarray for the immunohistochemical profiling of melanoma

Tissue microarray technology allows high throughput profiling of cancer specimens by immunohistochemical staining. Protein expression varies throughout tumour specimens resulting in heterogeneous staining patterns, which has led to doubts as to the accuracy of tissue microarray. In an effort to validate the use of tissue microarray for melanoma immunohistochemical investigation, a study was conducted comparing the concordance of MCAM staining between whole tumour specimens and tissue microarray core biopsies. Data on full tissue sections were compared with the results of one to four 0.6 mm core biopsies per tumour on a tissue array. It was revealed that concordance of tissue array core biopsies in quadruplicate compared with full-section analysis for the expression and intensity of expression of MCAM.     

大鼠大肠癌模型建立及其基因表达谱初步研究

目的建立实验性大肠癌动物模型,利用基因芯片技术筛选大肠癌发病进程中差异表达基因,探讨大肠癌发生发展过程中可能的信号通路。方法利用二甲肼诱发大鼠大肠癌,收集4例大鼠中的息肉、腺瘤、癌组织及对应正常组织,提取总RNA,应用大鼠全基因组表达谱芯片检测疾病及对应正常组织的基因表达情况,筛选不同组织之间的差异表达基因,以实时荧光定量PCR技术对基因芯片检测结果进行验证,最后利用生物信息技术对差异表达基因进行通路及功能归类。结果获得成功建模大鼠4只。基因芯片结果显示,息肉组织、腺瘤组织及大肠癌组织与正常大肠黏膜组织相比,差异表达基因分别为493、3 472及3 060个。大肠癌序列相关基因在3种病理组织中的表达均发生不同程度的变化。通路分析结果显示,在息肉组织中,细胞因子-细胞因子受体相互作用通路相关基因变化最为显著,P=5.62E-08;在腺瘤组织中,嘌呤代谢通路相关基因变化最为显著,P=7.73E-30;而在大肠癌组织中,核糖体通路相关基因变化最为显著,P=1.40E-27。功能分析结果显示,生物过程功能类归属基因表达变化最多,分子功能类基因其次,而细胞组分类基因最少。RT-PCR验证结果与芯片结果完全相符。结论在成功建立大肠癌动物模型的基础上,通过芯片技术筛选和鉴定了大肠癌疾病发生及发展相关基因,建立了肠息肉、腺瘤及大肠癌组织基因表达谱,为探寻大肠癌早期发病机制及基因靶向治疗提供了分子依据。     

Gene expression patterns and profile changes pre- and post-erlotinib treatment in patients with metastatic breast cancer.

PURPOSE: To delineate gene expression patterns and profile changes in metastatic tumor biopsies at baseline and 1 month after treatment with the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor erlotinib in patients with metastatic breast cancer. EXPERIMENTAL DESIGN: Patients were treated with 150 mg of oral erlotinib daily. Gene expression profiles were measured with Affymetrix U133A GeneChip and immunohistochemistry was used to validate microarray findings. RESULTS: Estrogen receptor (ER) status by immunohistochemistry is nearly coincided with the two major expression clusters determined by expression of genes using unsupervised hierarchical clustering analysis. One of 10 patients had an EGFR-positive tumor detected by both microarray and immunohistochemistry. In this tumor, tissue inhibitor of metalloproteinases-3 and collagen type 1 alpha 2, which are the EGF-down-regulated growth repressors, were significantly increased by erlotinib. Gene changes in EGFR-negative tumors are those of G-protein-linked and cell surface receptor-linked signaling. Gene ontology comparison analysis pretreatment and posttreatment in EGFR-negative tumors revealed biological process categories that have more genes differentially expressed than expected by chance. Among 495 gene ontology categories, the significant differed gene ontology groups include G-protein-coupled receptor protein signaling (34 genes, P = 0.002) and cell surface receptor-linked signal transduction (74 genes, P = 0.007). CONCLUSIONS: ER status reflects the major difference in gene expression pattern in metastatic breast cancer. Erlotinib had effects on genes of EGFR signaling pathway in the EGFR-positive tumor and on gene ontology biological process categories or genes that have function in signal transduction in EGFR-negative tumors.     

Predicting response to primary chemotherapy: gene expression profiling of paraffin-embedded core biopsy tissue

Purpose Primary chemotherapy provides an ideal opportunity to correlate gene expression with response to treatment. We used paraffin-embedded core biopsies from a completed phase II trial to identify genes that correlate with response to primary chemotherapy. Patients and Methods Patients with newly diagnosed stage II or III breast cancer were treated with sequential doxorubicin 75 mg/M2 q2 wks × 3 and docetaxel 40 mg/M2 weekly × 6; treatment order was randomly assigned. Pretreatment core biopsy samples were interrogated for genes that might correlate with pathologic complete response (pCR). In addition to the individual genes, the correlation of the Oncotype DX Recurrence Score with pCR was examined. Results Of 70 patients enrolled in the parent trial, core biopsies samples with sufficient RNA for gene analyses were available from 45 patients; 9 (20%) had inflammatory breast cancer (IBC). Six (14%) patients achieved a pCR. Twenty-two of the 274 candidate genes assessed correlated with pCR (p < 0.05). Genes correlating with pCR could be grouped into three large clusters: angiogenesis-related genes, proliferation related genes, and invasion-related genes. Expression of estrogen receptor (ER)-related genes and Recurrence Score did not correlate with pCR. In an exploratory analysis we compared gene expression in IBC to non-inflammatory breast cancer; twenty-four (9%) of the genes were differentially expressed (p < 0.05), 5 were upregulated and 19 were downregulated in IBC. Conclusion Gene expression analysis on core biopsy samples is feasible and identifies candidate genes that correlate with pCR to primary chemotherapy. Gene expression in IBC differs significantly from noninflammatory breast cancer.     

Gene expression profiling identifies molecular subtypes of inflammatory breast cancer

Breast cancer is a heterogeneous disease. Comprehensive gene expression profiles obtained using DNA microarrays have revealed previously indistinguishable subtypes of noninflammatory breast cancer (NIBC) related to different features of mammary epithelial biology and significantly associated with survival. Inflammatory breast cancer (IBC) is a rare, particular, and aggressive form of disease. Here we have investigated whether the five molecular subtypes described for NIBC (luminal A and B, basal, ERBB2 overexpressing, and normal breast-like) were also present in IBC. We monitored the RNA expression of approximately 8,000 genes in 83 breast tissue samples including 37 IBC, 44 NIBC, and 2 normal breast samples. Hierarchical clustering identified the five subtypes of breast cancer in both NIBC and IBC samples. These subtypes were highly similar to those defined in previous studies and associated with similar histoclinical features. The robustness of this classification was confirmed by the use of both alternative gene set and analysis method, and the results were corroborated at the protein level. Furthermore, we show that the differences in gene expression between NIBC and IBC and between IBC with and without pathologic complete response that we have recently reported persist in each subtype. Our results show that the expression signatures defining molecular subtypes of NIBC are also present in IBC. Obtained using different patient series and different microarray platforms, they reinforce confidence in the expression-based molecular taxonomy but also give evidence for its universality in breast cancer, independently of a specific clinical form.     

In vivo gene expression profile analysis of human breast cancer progression

The development and use of molecular-based therapy for breast cancer and other human malignancies will require a detailed molecular genetic analysis of patient tissues. The recent development of laser capture microdissection and high density cDNA arrays now provides a unique opportunity to generate gene expression profiles of cells from various stages of tumor progression as it occurs in the actual neoplastic tissue milieu. We report the combined use of laser capture microdissection and high-throughput cDNA microarrays to monitor in vivo gene expression levels in purified normal, invasive, and metastatic breast cell populations from a single patient. These in vivo gene expression profiles were verified by real-time quantitative PCR and immunohistochemistry. The combined use of laser capture microdissection and cDNA microarray analysis provides a powerful new approach to elucidate the in vivo molecular events surrounding the development and progression of breast cancer and is generally applicable to the study of malignancy.     

Microarray analysis in clinical oncology: pre-clinical optimization using needle core biopsies from xenograft tumors

Background

DNA microarray profiling performed on clinical tissue specimens can potentially provide significant information regarding human cancer biology. Biopsy cores, the typical source of human tumor tissue, however, generally provide very small amounts of RNA (0.3–15 μg). RNA amplification is a common method used to increase the amount of material available for hybridization experiments. Using human xenograft tissue, we sought to address the following three questions: 1) is amplified RNA representative of the original RNA profile? 2) what is the minimum amount of total RNA required to perform a representative amplification? 3) are the direct and indirect methods of labeling the hybridization probe equivalent?

Methods

Total RNA was extracted from human xenograft tissue and amplified using a linear amplification process. RNA was labeled and hybridized, and the resulting images yielded data that was extracted into two categories using the mAdb system: "all genes" and "outliers". Scatter plots were generated for each slide and Pearson Coefficients of correlation were obtained.

Results

Results show that the amplification of 5 μg of total RNA yields a Pearson Correlation Coefficient of 0.752 (N = 6,987 genes) between the amplified and total RNA samples. We subsequently determined that amplification of 0.5 μg of total RNA generated a similar Pearson Correlation Coefficient as compared to the corresponding original RNA sample. Similarly, sixty-nine percent of total RNA outliers were detected with 5 μg of amplified starting RNA, and 55% of outliers were detected with 0.5 μg of starting RNA. However, amplification of 0.05 μg of starting RNA resulted in a loss of fidelity (Pearson Coefficient 0.669 between amplified and original samples, 44% outlier concordance). In these studies the direct or indirect methods of probe labeling yielded similar results. Finally, we examined whether RNA obtained from needle core biopsies of human tumor xenografts, amplified and indirectly labeled, would generate representative array profiles compared to larger excisional biopsy material. In this analysis correlation coefficients were obtained ranging from 0.750–0.834 between U251 biopsy cores and excised tumors, and 0.812–0.846 between DU145 biopsy cores and excised tumors.

Conclusion

These data suggest that needle core biopsies can be used as reliable tissue samples for tumor microarray analysis after linear amplification and either indirect or direct labeling of the starting RNA.

     

Diagnostic accuracy of large-core needle biopsy for nonpalpable breast disease: a meta-analysis

For the evaluation of non-palpable lesions of the breast, image-guided large-core needle biopsies are increasingly replacing needle-localized open breast biopsies. In this study, the diagnostic accuracy of this minimally invasive technique was evaluated by reviewing the available literature. Five cohort studies were included in a meta-analysis. Sensitivity rate, histological agreement between needle biopsy and subsequent surgery or long-term mammographic follow-up and clinical consequences for different disease prevalences were assessed. The sensitivity rate of large-core needle biopsy for the diagnosis of breast cancer was high (97%). The reclassified agreement rate between core biopsy and subsequent surgical biopsy or long-term mammographic follow-up was also high (94%). In case of 20% breast cancer prevalence among women referred after screening (as in the US), the risk of breast cancer despite benign large-core needle biopsy result is less than 1%. In European countries, however, prevalence of breast cancer among referred women is 60-70%. This would result in a risk of breast cancer despite benign large-core needle biopsy result of 4-6%. The results of this meta-analysis indicate that the image guided large-core needle biopsy is a promising alternative for the needle localized breast biopsy. However, additional research is needed to explore the limiting factors of the technique. Without such detailed knowledge, a benign histological diagnosis on large-core needle biopsy in countries with high prevalence of malignancy among referred women should be interpreted with caution.     

Classification of human tumors using gene expression profiles obtained after microarray analysis of fine-needle aspiration biopsy samples

BACKGROUND: Gene expression profiling using gene-discovery, high-density microarray technologies is a powerful tool. One potential application is the development of tumor classifiers that predict the site of origin. For this technology to be relevant, however, it must be applicable to tumor biopsy samples, which most often are fine-needle aspiration biopsy (FNAB) samples. METHODS: Surgically resected tumors were sampled by FNAB using different gauge needles. A portion of the excised tumor was also collected. RNA samples were extracted using standard techniques and the quality and quantity of the RNA samples were measured for each sample. Thirteen representative FNAB samples and two representative tissue samples were submitted for microarray analysis and then subjected to a tumor classifier. RESULTS: Fourteen of 18 samples analyzed for quantity and quality of RNA yielded an adequate amount of RNA (> 1 microg total RNA). Tumor type contributed to the RNA yield because one of the four inadequate samples was retrieved from a patient with lobular carcinoma of the breast and the other three samples were retrieved from patients with retroperitoneal sarcomas. Of the 13 samples submitted for microarray analysis, 9 were classified correctly as to tumor type using a tissue-based tumor classifier. CONCLUSIONS: The authors demonstrated that FNABs reproducibly obtained an adequate amount of RNA for microarray analysis when a standardized collection procedure was used. Furthermore, the samples generated interpretable gene expression profiles that could be matched accurately with a tumor classifier established on tissue specimens. The current study showed that FNAB produced adequate material for microarray analysis when utilizing a standardized collection procedure.