Human Genome Survey Microarray Research Articles

Abstract P3-04-03: A seven-gene profile predicting benefit of postmastectomy radiotherapy independently of nodal status in high risk breast cancer

Abstract Background: Recommendations for postmastectomy radiotherapy (PMRT) are well established in patients estimated to have a high risk of loco-regional recurrence (LR) (e.g. tumor size > 5 cm or ≥ 4 positive lymph nodes). International recommendations for patients with 1–3 positive lymph nodes suggest that PMRT should be restricted to younger patients and patients with other poor prognostic features. However, large randomized trials, including the DBCG82 trials (Danish Breast Cancer Cooperative Group), have previously shown a substantial overall survival benefit after PMRT in patients with low risk of LR, and shown that the largest translation of LR reduction into breast cancer mortality reduction occurs within the most favorable prognosis group. Our hypothesis is that a more refined partitioning of patients likely to benefit from PMRT can be established through identification of genes whose transcription interacts with PMRT to modify the hazard of LR. Material and methods: The DBCG82bc cohort constitutes high risk patients (tumor size > 5 cm and/or positive nodes and/or invasion in skin or pectoral fascia) diagnosed between 1983–89, treated with mastectomy and partial axillary lymph nodes dissection and randomized to +/− PMRT. From 267 DBCG82bc patients, frozen tumor samples were available. Whole genome arrays (Applied Biosystem Human Genome Survey Microarray v2.0®, Applied Biosystem, Foster City, USA) were successful in 195 samples. Genes, whose expression levels interacted with PMRT on the association with LR, were identified through a two step Cox Proportional Hazard model with lasso penalty. 11 node negative patients were excluded from the subsequent analysis of 184 node positive patients (1–3 pos. nodes: 102 pts, ≥ 4 pos. nodes: 82 pts). Results: Seven genes were identified whose expression interact with the effect of PMRT, and a specialized index was generated based on the expression levels of these genes. Patients were ranked according to the size of the index, and divided into quartiles with 25% of the patients designated as having a “high index” and 75% a “low index”. Among patients not receiving PMRT, a low index was in both nodal groups (1–3 vs. ≥ 4 positive nodes) associated with a significantly higher risk of LR compared to patients with a high index. In both nodal groups, PMRT significantly reduced the risk of LR in patients with a low index; equalizing the risk to patients with a high index, who showed no additional LR reduction by PMRT. In the group of 1–3 positive nodes, PMRT raised the local control rate (LCR) after 10 years from 47.5 % to 91.8% in the low index group (p = 0.0001), whereas the change in the high index group was non-significant (92.3% vs. 100.0%). In the group of patients with ≥ 4 positive nodes, PMRT raised LCR from 16.6% to 80.0% in the low index group (p = 0.0001), and no effect on LCR was seen in the high index group (83.3% vs. 87.5%, n.s.). Conclusion: A seven gene-profile attaining prognostic and predictive impact, irrespective of number of positive nodes, was identified. The profile allowed the identification of 25% of the patients not showing any additional benefit from PMRT in terms of LR. The gene-profile may provide a method to identify patients expected to benefit from PMRT. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-04-03.

Investigation of gene expression profiles before and after embryonic genome activation and assessment of functional pathways at the human metaphase II oocyte and blastocyst stage

To compare the oocyte versus the blastocyst transcriptome and provide data on molecular pathways before and after embryonic genome activation. Prospective laboratory research study. An IVF clinic and a specialist preimplantation genetics laboratory. Couples undergoing or having completed IVF treatment donating surplus oocytes or cryopreserved blastocysts after patient consent. Sets of pooled metaphase II (MII) oocytes or blastocysts were processed for RNA extraction, RNA amplification, and analysis with the use of the Human Genome Survey Microarrays v2.0 (Applied Biosystems). Association of cell type and gene expression profile. Totals of 1,909 and 3,122 genes were uniquely expressed in human MII oocytes and human blastocysts respectively, and 4,910 genes were differentially expressed between the two sample types. Expression levels of 560 housekeeping genes, genes involved in the microRNA processing pathway, as well as hormones and hormone receptors were also investigated. The lists of genes identified may be of use for understanding the processes involved in early embryo development and blastocyst implantation, and for identifying any dysregulation leading to infertility.

Absence of Transcriptomic Signature of Response to Chemotherapy in Metastatic Colorectal Carcinoma Patients

Tumor gene-expression profiling may define signatures capable of discriminating between responders and nonresponders to chemotherapy. Fifty seven metastatic colorectal cancer patients were prospectively included and 40 tumors were analyzed. Patients were treated in first line with 5-fluorouracil associated with irinotecan or oxaliplatin. Response was evaluated using WHO criteria every 2 months after chemotherapy. Gene-expression profiling was performed using Applied Biosystems microarrays (Human Genome Survey Microarray v2.0; Paris, France). Data were analyzed using Bioconductor packages. Differential-expression analysis was performed by fitting a linear model. Moderated t-statistics were computed and p-values were adjusted for false-discovery rate. Pearson correlations tests were evaluated between gene expression and progression-free and overall survival. Nonsupervised analysis did not show any clustering of expression levels according to treatment response. Supervised analysis compared expression levels between responders and nonresponders, within each treatment group and independently from treatment. No genes were identified as differentially expressed at a p-value of 10(-3) and false-discovery rate of 30%. No correlation between expression levels and survival data was found. These negative results show that the determinants of response to chemotherapy should be sought not only in the tumor characteristics, but also among the processes leading to drug availability to the tumor.

Abstract 2606: Absence of transcriptomic signature of response to chemotherapy in metastatic colorectal carcinoma patients

Abstract Using whole genome tumour gene expression profiling in patients treated for metastatic colorectal cancer, we attempted to define a signature able to discriminate between responders and non-responders to first-line chemotherapy. A total of 42 patients were prospectively included between 2003 and 2006. They all suffered from histologically proven colorectal adenocarcinoma and were metastatic at the time of diagnosis. All of them gave informed consent to the study. They were treated in first line with 5-fluorouracil (according to the de Gramont regimen in most cases) associated either with irinotecan (FolFiri regimen, 180 mg/m2 every two weeks, 22 pts) or with oxaliplatin (FolFox 4 regimen, 85 mg/m2, 20 pts) according to physician's decision. Response was evaluated using WHO criteria every 2 months after initiation of chemotherapy and patients were assigned to complete response (1 pt), partial response (24 pts), stable disease (13 pts) or progressive disease (4 pts). From a tumour sample obtained at surgery (18 pts) or by biopsy (24 pts) and kept in RNA later, we extracted and purified total RNA. Gene expression profiling was performed by Cogenics using microarrays from Applied Biosystems (Human Genome Survey Microarray v2.0). We used three different methods for supervised data analysis: analysis of variance, SAM (statistical analysis of microarrays) and Limma (Bioconductor). We compared responders to non-responders in the whole set of patients and in the subsets of patients who received either irinotecan or oxaliplatin. With none of the three methods we were able to obtain a stable signature, i.e. a group of genes consistently associated with response to chemotherapy. On the contrary, we obtained very homogeneous gene expression profiles which were even difficult to clusterise using a non-supervised approach. These negative results show that the determinants of response to chemotherapy should be sought not only in the tumour molecular characteristics, but also among the processes leading to drug availability to the tumour (drug dose, drug disposition, etc.). Especially, the relatively low dose of irinotecan which is of standard use in Europe does not allow to recruit all the responders who could be identified at higher dosage, given the sharp dose-efficacy curve that has been identified long ago for this drug. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2606.

Gene Expression Profiling for Therapy Prediction in a Breast Cancer Neoadjuvant Therapy Study Applying Docetaxel/Epirubicin/Cyclophosphamide.

Abstract Background: Gene expression profiling is a powerful tool to identify markers associated with clinical and therapy outcome of cancer patients. Prediction of the response to neoadjuvant regimens remains a persistent challenge. Aim of this ongoing study is the development of a gene set predicting the response of the tumor to a taxane- anthracycline based neoadjuvant chemotherapy.Material and Methods: Microarray expression profiling was performed on biopsy samples from patients before treatment using Human Genome Survey microarrays (HGSM). The protocol for a phase II study was elaborated for the treatment of breast cancer patients suffering from a primary tumor 1.5 cm or inflammatory breast cancer with Docetaxel/Epirubicin/Cyclophosphamide (TEC) prior to surgical treatment. The study was approved by the local ethical committee and all patients signed an informed consent.Results: Overall 80 patients have been enrolled in the presented study. High quality gene expression data were available from 58 patients. Of these patients 21 responded to the TEC regimen (pCR or MIB1 expressing cells in the residual tumor <= 5% and decrease of Mib1-expression >= 20%). Based on the gene-expression profile we were able to identify a preliminary gene set of 150 genes which allows us to separate responding tumors from the non responding ones based on their gene expression profile. A comparable separation of the groups could not achieved by established tumor markers, e.g. ER, PgR, HER2, uPA etc. which are measured simultaneously on the HGSM. Among the genes distinguishing the two groups several genes normally expressed in mononuclear blood cells were identified, pointing to the presence of tumor infiltrating leukocytes, predominantly in the tumors responding the TEC regimen. The presence of these cells has already been verified in a subset of the samples.Conclusion: We identified a gene set which allows to select patients who will benefit from neoadjuvant chemotherapy. Furthermore at least in the so far investigated samples tumor infiltrating leukocytes are significantly more often found in tumors which respond to a taxane- anthracycline based neoadjuvant chemotherapy. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2039.

Prokineticin-1 (PROK1) modulates interleukin (IL)-11 expression via prokineticin receptor 1 (PROKR1) and the calcineurin/NFAT signalling pathway

Prokineticin-1 (PROK1) is a multifunctional secreted protein which signals via the G-protein coupled receptor, PROKR1. Previous data from our laboratory using a human genome survey microarray showed that PROK1–prokineticin receptor 1 (PROKR1) signalling regulates numerous genes important for establishment of early pregnancy, including the cytokine interleukin (IL)-11. Here, we have shown that PROK1–PROKR1 induces the expression of IL-11 in PROKR1 Ishikawa cells and first trimester decidua via the calcium–calcineurin signalling pathway in a guanine nucleotide-binding protein (Gq/11), extracellular signal-regulated kinases, Ca2+ and calcineurin–nuclear factor of activated T cells dependent manner. Conversely, treatment of human decidua with a lentiviral miRNA to abolish endogenous PROK1 expression results in a significant reduction in IL-11 expression and secretion. Importantly, we have also shown a regulatory role for the regulator of calcineurin 1 isoform 4 (RCAN1-4). Overexpression of RCAN1-4 in PROKR1 Ishikawa cells using an adenovirus leads to a reduction in PROK1 induced IL-11 indicating that RCAN1-4 is a negative regulator in the calcineurin-mediated signalling to IL-11. Finally, we have shown the potential for both autocrine and paracrine signalling in the human endometrium by co-localizing IL-11, IL-11Rα and PROKR1 within the stromal and glandular epithelial cells of non-pregnant endometrium and first trimester decidua. Overall we have identified and characterized the signalling components of a novel PROK1–PROKR1 signalling pathway regulating IL-11.

Genome-based expression profiles study for the pathogenesis of pelvic organ prolapse: an array of 33 genes model

To explore the potential molecular mechanisms contributing to the pathogenesis of pelvic organ prolapse (POP) with the aid of high-density oligonucleotide microarrays. We compared microarray gene expression profiles in pelvic connective tissue from women with POP and nonprolapse controls. The round ligament and uterosacral ligament tissues were removed from each subject at the time of laparoscopic hysterectomy. RNA was then extracted, and all labeled samples were hybridized to ABI Human Genome Survey Microarray version 2.0 (Applied Biosystems, CA, USA). The Mahalanobis distance in hierarchical cluster analysis revealed a model of 33 genes, which contained high expressions of round and uterosacral ligaments from women with POP. According to gene ontology, the expressions of mitochondrial genes encoding ribosomal protein were upregulated. Genes involved in potential interactions with mitochondrial electron transport, nucleosome assembly, cell cycle, and apoptosis were also upregulated. As a result, defective mitochondrial translation caused by ribosomal protein contributes to the potential molecular etiology of POP. Such changes, jointly termed "remodeling of pelvic connective tissue", can constitute an important long-term consequence of POP. Our results support the use of genome-based expression profiling as a commonplace platform for diagnostic tests of POP.

Expression profiling of DNA repair genes in human oocytes and blastocysts using microarrays

The early preimplantation embryo relies on mRNA and protein from the oocyte to detect DNA damage and activate DNA repair, cell cycle arrest or apoptosis. Expression of some repair genes has been detected in mammalian oocytes and embryos; however, little is known about DNA repair gene expression in human blastocysts. In this study, DNA repair gene expression was investigated in human oocytes and blastocysts to identify the pathways involved at these stages and detect potential differences in repair mechanisms pre- and post-embryonic genome activation. Triplicate sets of pooled metaphase II oocytes or blastocysts were processed for analysis using the Human Genome Survey Microarrays V2.0 (Applied Biosystems). Of 154 DNA repair genes investigated, 109 were detected in blastocysts and 107 in oocytes. Among differentially expressed DNA repair genes, 40/55 (73%) had lower expression levels in blastocysts compared with oocytes (P < 0.05, fold change >3). Despite experimental limitations due to culture or freezing and thawing of samples, large numbers of repair genes were detected indicating that all DNA repair pathways are potentially functional in human oocytes and blastocysts. The higher mRNA level for most repair genes in oocytes compared with blastocysts ensures sufficient availability of template until embryonic genome activation.

Differential Placental Gene Expression in Severe Preeclampsia

We investigated the global placental gene expression profile in severe preeclampsia. Twenty-one women were randomly selected from 50 participants with uncomplicated pregnancies to match 21 patients with severe preeclampsia. A 30 K Human Genome Survey Microarray v.2.0 (Applied Biosystems) was used to evaluate the gene expression profile. After RNA isolation, five preeclamptic placentas were excluded due to poor RNA quality. The series composed of 37 hybridizations in a one-channel detection system of chemiluminescence emitted by the microarrays. An empirical Bayes analysis was applied to find differentially expressed genes. In preeclamptic placentas 213 genes were significantly (fold-change ≥ 2 and p ≤ 0.01) up-regulated and 82 were down-regulated, compared with normal placentas. Leptin (40 fold), laeverin (10 fold), different isoforms of β-hCG (3–6 fold), endoglin (4 fold), FLT1 (3 fold) and FLT4 (2 fold) were up-regulated. PDGFD was down-regulated (2 fold). Several differentially expressed genes were associated with Alzheimer disease, angiogenesis, Notch-, TGFβ- and VEGF-signalling pathways. Sixteen genes best discriminated preeclamptic from normal placentas. Comparison between early- (<34 weeks) and late-onset preeclampsia showed 168 differentially expressed genes with oxidative stress, inflammation, and endothelin signalling pathways mainly involved in early-onset disease. Validation of the microarray results was performed by RT-PCR, quantitative urine hCG measurement and placental histopathologic examination. In summary, placental gene expression is altered in preeclampsia and we provide a comprehensive list of the differentially expressed genes. Placental gene expression is different between early- and late-onset preeclampsia, suggesting differences in pathophysiology.

The Role of Zinc Finger Protein 521/Early Hematopoietic Zinc Finger Protein in Erythroid Cell Differentiation

ZNF521 (zinc finger protein 521) is a transcription factor with an N-terminal transcriptional repressor motif and 30 zinc finger domains. Although a high expression level of ZNF521 in human CD34+ progenitors and hematopoietic malignancies has been demonstrated, the functional role of ZNF521 in hematopoietic cell differentiation has not been clarified. In this study, we analyzed the role of ZNF521 in erythroid cell differentiation using the short hairpin RNA (shRNA)-mediated gene silencing method. Down-regulation of ZNF521 mediated by transient expression of shRNA for ZNF521 resulted in increased synthesis of hemoglobin in K562 and HEL cell lines as compared with control cells. K562-derived clones in which ZNF521 was constitutively silenced by shRNA also showed marked synthesis of hemoglobin and an increased expression level of glycophorin A. Since GATA-1 is the key regulator of erythroid differentiation, the effect of ZNF521 on transcription activity of GATA-1 was analyzed using a luciferase assay. GATA-1 activity was markedly inhibited by ZNF521 in a dose-dependent manner. Deletion analysis of ZNF521 showed that the repressive effect requires an N-terminal repression motif. Furthermore, the direct interaction of ZNF521 with GATA-1 was demonstrated. These results indicate that ZNF521 modulates erythroid cell differentiation through direct binding with GATA-1.

A gene signature completely identifies metastasized testis tumors

22144 Background: Selection of gene targets to distinguish metastasized from non-metastasized seminoma. Methods: Total RNA was isolated from metastasized seminoma (n=10, T1–3N0M0 to T1N1–2M0), non-metastasized seminoma (n=21) and corresponding normal tissues. The RNA from 10 biopsies of each tissue type was pooled and separately hybridized on four whole genome microarrays (Human Genome Survey Microarray V2.0, Applied Biosystem) for screening purposes. 92 selected gene candidates were then quantitatively examined using RTQ-PCR. Results: Agreement in gene expression was 88% between the whole genome microarrays and RTQ-PCR. Metastasized seminoma showed 1,912 upregulated and 2,179 downregulated genes with ≥ 2-fold differences in gene expression as compared to non-metastasized seminoma. The number of upregulated genes coding for immunity and defense, cell signaling and developmental processes were significantly overrepresented. RTQ-PCR showed that mean gene expression values of the 92 genes in general were significantly reduced in metastasized compared to non-metastasized seminoma. The presence of metastases could be completely predicted (100% specificity) based on a 85-gene expression signature by using Logistic Regression. Accompanied sensitivity and accuracy of the 10-fold crossvalidation model were 77.8% and 84.2%, respectively. Conclusions: A logistic regression model using the 85 gene expression signature allowed complete identification of metastasized seminoma. No significant financial relationships to disclose.

Microarray analysis identifies a common set of cellular genes modulated by different HCV replicon clones

BackgroundHepatitis C virus (HCV) RNA synthesis and protein expression affect cell homeostasis by modulation of gene expression. The impact of HCV replication on global cell transcription has not been fully evaluated. Thus, we analysed the expression profiles of different clones of human hepatoma-derived Huh-7 cells carrying a self-replicating HCV RNA which express all viral proteins (HCV replicon system).ResultsFirst, we compared the expression profile of HCV replicon clone 21-5 with both the Huh-7 parental cells and the 21-5 cured (21-5c) cells. In these latter, the HCV RNA has been eliminated by IFN-α treatment. To confirm data, we also analyzed microarray results from both the 21-5 and two other HCV replicon clones, 22-6 and 21-7, compared to the Huh-7 cells. The study was carried out by using the Applied Biosystems (AB) Human Genome Survey Microarray v1.0 which provides 31,700 probes that correspond to 27,868 human genes. Microarray analysis revealed a specific transcriptional program induced by HCV in replicon cells respect to both IFN-α-cured and Huh-7 cells. From the original datasets of differentially expressed genes, we selected by Venn diagrams a final list of 38 genes modulated by HCV in all clones. Most of the 38 genes have never been described before and showed high fold-change associated with significant p-value, strongly supporting data reliability. Classification of the 38 genes by Panther System identified functional categories that were significantly enriched in this gene set, such as histones and ribosomal proteins as well as extracellular matrix and intracellular protein traffic. The dataset also included new genes involved in lipid metabolism, extracellular matrix and cytoskeletal network, which may be critical for HCV replication and pathogenesis.ConclusionOur data provide a comprehensive analysis of alterations in gene expression induced by HCV replication and reveal modulation of new genes potentially useful for selection of antiviral targets.

MRNA transcript profiling of human oocytes at different maturational stages and after in vitro maturation

To catalogue and compare genes expressed in human oocytes at germinal vesicle (GV) or metaphase II (MII) stage and after in vitro maturation (IVM). Descriptive research assessing unfertilized oocytes by microarray. GV, in vivo matured MII or IVM-MII oocytes were collected and processed rapidly. RNA was extracted and amplified using a 2-round in vitro transcription procedure, then labeled and hybridized to Applied Biosystems Human Genome Survey Microarrays. This array interrogates 29,098 genes. Up- or down-regulated genes were classified according to the molecular function, biological process, or pathway in which they participate. Statistical analysis was employed to compare each class of genes between samples. The in vitro transcription protocol generated an average of 12 μg of RNA per sample. GV, in vivo matured-MII and IVM-MII oocytes expressed 14,188, 16,402 and 18,299 genes, respectively. There was extensive overlap in the genes expressed by each group, but also some significant differences. Samples showing the least similarity in expression profile were GV and in vivo MII oocytes. IVM-MIIs also displayed significant differences in expression when compared to in vivo MII oocytes, indicating that the process of in vitro maturation does not perfectly mimic that achieved in vivo. A disproportionate number of genes for storage proteins displayed down-regulation in IVM-MII oocytes (relative to in vivo MII). A reduction in expression was also seen for genes involved in processes of homeostasis. Significant up-regulation of gene expression for the ubiquitin proteasome pathway was observed in both IVM-MII and GV samples. This study provided a detailed insight into gene expression of human oocytes, assessing virtually every gene in the genome. GV oocytes were highly active for processes related to transcription, RNA processing and early stages of protein synthesis, whereas MII oocytes were active for pathways involved in processing of mature proteins. The results suggest that although IVM-MII oocytes closely resemble in vivo-MII oocytes for pathways related to nuclear maturity, a number of those associated with cytoplasmic functions continue to be expressed in a GV-like manner. Additionally, IVM-MII oocytes have significant differences in the expression of genes related to cellular storage and homeostasis. The pathways abnormally expressed during IVM highlight specific deficiencies in culture media and point the way to further optimization of IVM protocols.

Semi-quantitative gene expression profiling for therapy prediction in a breast cancer neoadjuvant therapy study applying docetaxel/epirubicin/cyclophosphamide

21144 Background: More than 28000 curated human genes can be analyzed semi-quantitatively using a chemiluminescent detection technology and 60mer oligonucleotides on a Human Genome Survey Microarray (HGSM, Applied Biosytems). Methods: HGSM expression profiling was performed on biopsy samplesfrom a setting of patients under neoadjuvant treatment. The protocol for a phase II study was elaborated for the treatment of breast cancer patients suffering from a primary tumor &gt; 1,5 cm or inflammatory breast cancer with Docetaxel / Epirubicin/Cyclophosphamide (TEC) prior to surgical treatment. The study was approved by the local ethical committee and 80 patients will be included into the study after written informed consent. 40 patients have been already included in the ongoing study. In this prospective study a biopsy from the tumor is taken before chemotherapy. Therefore, success of treatment is detectable directly at the operated residual tumor. Our results demonstrate a low rate of false-positives (1.2%), a high specificity and quantification accuracy of HGSM system. Comparison of data from HGSM and RT-PCR obtained on mRNA from fresh frozen tissue resulted a Pearson-correlation of 0.92 to 0.63 for the breast cancer genes. Results: Tumour response (pCR, pPR) of more than 70 % can be achieved using neoadjuvant TEC- regimen. 25 % of pCR in this study is comparable with data of other published neoadjuvant trails. First expression profiling results are obtainable showing that a subset of 148 genes indicates patients with complete remission (pCR, no detectable tumor at end of chemotherapy), partial remission (pPR) and progressive disease (pPD). Remarkable, that the expression profile clearly separated pCR and pPD tumors whereas pPR tumors presented with a closer relationship to pPD tumors than to pCR tumors but displayed small specific subprofiles. A comparable separation of the groups could not achieved by established tumor markers, e.g. ER, PgR, HER2, uPA etc. which are measured simultaneously on the HGSM. Conclusions: HGSM expression profiling is promising to have the potential to figure out genes that are related to cancer progression and chemotherapy resistance, especially in PST. No significant financial relationships to disclose.

Cholinergic Regulation of Morphine Release from Human White Blood Cells: Evidence for a Novel Nicotinic Receptor via Pharmacological and Micro Array Analysis

Recent work from our laboratory has demonstrated that human white blood cells make morphine and that substances of abuse, i.e. nicotine, alcohol and cocaine have the ability to release this endogenous substance, suggesting a common mechanism of action. We now demonstrate that the nicotinic process is more complex than formerly envisioned. The incorporation rate of 125I-labeled morphine into PMN and MN are 7.85+/-0.36%, 1.42+/-0.19%, respectfully, suggesting in MN this process is of low activity. Separate incubations of PMN with varying concentrations of nicotine or the nicotine agonist epibatidine resulted in a statistically significant enhancement of 125I-trace labeled morphine released into the extracellular medium. In order to ascertain the specificity of the nicotine stimulated morphine release the following experiments were performed. Co-incubation of hexamethonium dichloride (5 microg/ml and at 10 microg/ml), which preferentially blocks nicotinic receptors at autonomic ganglia, with nicotine, exerted a very weak inhibitory effect. Co-incubation of alpha-BuTx or atropine or chlorisondamine diiodide or dihydro-Beta-erythroidine hydrobromide, an alpha4Beta2 receptor antagonist, did not block nicotine induced morphine release alone or in combination, suggesting either the response was not specific or it was mediated by a novel nicotinic receptor. Human leukocyte total RNA isolated from whole blood were analyzed, using the Human Genome Survey microarray (Applied Biosystems), for cholinergic receptor expression. PMN nicotinic receptor gene expression was present and contained numerous variants (eight). The number of variants suggests that indeed a novel nicotinic receptor may be mediating this effect, while simultaneously demonstrating the significance of the cholinergic receptor expression in these immune cells.

Human Platelet-Derived Factors Regulate Mesenchymal Stem Cell Gene Expression.

The use of animal-derived products during human stem cell processing bears the evident risk of xenogeneic prion, virus, or zoonose contamination. Human platelet lysate (HPL) has recently been recognized as a rich source of cytokines and growth factors with the potential to replace fetal bovine serum (FBS) during ex vivo stem cell manipulation. In this study we compared the gene expression profile of human multipotent mesenchymal stromal/stem cells (MSC) during ex vivo expansion for clinical applications under the aegis of either FBS or HPL.The Applied Biosystems 1700 Expression Array System was used for full genome expression profiling of MSC after a 12–14 day expansion period in a previously optimized low density expansion system. Data have been obtained from biological as well as technical replicates. A starting amount of 40μg total RNA was directly labeled and DIG-labeled cDNA was hybridized to Human Genome Survey Microarray V2.0. Attribution of regulated genes to biological processes and pathways was done using the PANTHER® db analysis software.We identified more than 300 genes that are differentially regulated upon culture of MSC in HPL compared to FBS. Biological processes specifically activated in HPL culture include mesoderm development, cell cycle control, hematopoiesis and angiogenesis which interestingly correspond to a considerable proportion of the regenerative function of MSC. In contrast, processes related to cell adhesion and adhesion-mediated signaling, cell structure, cell motility and cell communication are significantly upregulated in MSC after FBS in comparison to HPL culture.Replacing FBS with HPL not only avoids bovine prion, viral and zoonose contamination of MSC for clinical use. The tightly regulated gene expression profiles under the aegis of human growth factors and cytokines provided by HPL may even help to develop new stem cell therapy strategies.

Therapy prediction in a breast cancer primary systemic chemotherapy (PST) study applying docetaxel/epirubicin/cyclophosphamide (TEC) by gene expression profiling

10049 Background: Currently there are no tests to assist in selecting the optimal PST regimen for breast cancer patients. Primary study goals of this prospective, single-armed multicentric investigation are pathologically confirmed tumor response and the rate of breast conserving therapy (BCT). Secondary goals are to find histopathologic and gene profiling patterns best correlating with tumor remission in a taxane- anthracycline based neoadjuvant setting as well as to evaluate cytostatic toxicity and quality of life. Methods: In this phase II study of totally 40 eligible patients with invasive breast cancer Human Genome Survey Microarray (HGSM) expression profiling is performed on jet-biopsy sample basis. The protocol was elaborated for the treatment of patients suffering from a primary tumor with 6 cycles of TEC (3-weekly) prior to the surgical treatment. The selection of predictor genes was done with BRB-ArrayTools Version 3.3 using a model based on the Compound Covariate Predictor, Diagonal Linear Discriminant Analysis, Nearest Neighbor Classification, and Support Vector Machines with linear kernel.We estimated the prediction error of each model using leave-one-out cross-validation (LOOCV) as described by Simon R. 2000 random permutations were used. Clustering was done using Cluster 3.0 and Java TreeView 1.0.12. Results: Tumor response (pCR, pPR) of more than 70% can be achieved using neoadjuvant TEC-regimen. 22% pCR (ypT0; ypN0) and 90% BCT in this study are comparable with data of other published PST trials. Preliminary expression profiling results reveal a subset of 148 genes that classifies all patients with a complete remission (pCR), in one cluster with a very closely related gene expression pattern (n=5; PPV = 100%). Furthermore 10 patients defined as responders due to selected MIB1-expression based criteria (expressing cells in the residual tumor ≤ 5% and a Δ MIB1-expression ≥ 20%) can be correctly classified in 9 of 10 cases. Comparable separation of the groups could not be achieved by established tumor factors. Conclusions: HGSM expression profiling is promising to have the potential to figure out genes that are related to chemotherapy response, especially in PST. No significant financial relationships to disclose.

Distinct molecular mechanisms underlying clinically relevant subtypes of breast cancer: gene expression analyses across three different platforms

BackgroundGene expression profiling has been used to define molecular phenotypes of complex diseases such as breast cancer. The luminal A and basal-like subtypes have been repeatedly identified and validated as the two main subtypes out of a total of five molecular subtypes of breast cancer. These two are associated with distinctly different gene expression patterns and more importantly, a significant difference in clinical outcome. To further validate and more thoroughly characterize these two subtypes at the molecular level in tumors at an early stage, we report a gene expression profiling study using three different DNA microarray platforms.ResultsExpression data from 20 tumor biopsies of early stage breast carcinomas were generated on three different DNA microarray platforms; Applied Biosystems Human Genome Survey Microarrays, Stanford cDNA Microarrays and Agilent's Whole Human Genome Oligo Microarrays, and the resulting gene expression patterns were analyzed. Both unsupervised and supervised analyses identified the different clinically relevant subtypes of breast tumours, and the results were consistent across all three platforms. Gene classification and biological pathway analyses of the genes differentially expressed between the two main subtypes revealed different molecular mechanisms descriptive of the two expression-based subtypes: Signature genes of the luminal A subtype were over-represented by genes involved in fatty acid metabolism and steroid hormone-mediated signaling pathways, in particular estrogen receptor signaling, while signature genes of the basal-like subtype were over-represented by genes involved in cell proliferation and differentiation, p21-mediated pathway, and G1-S checkpoint of cell cycle-signaling pathways. A minimal set of 54 genes that best discriminated the two subtypes was identified using the combined data sets generated from the three different array platforms. These predictor genes were further verified by TaqMan® Gene Expression assays.ConclusionWe have identified and validated the two main previously defined clinically relevant subtypes, luminal A and basal-like, in a small set of early stage breast carcinomas. Signature genes characterizing these two subtypes revealed that distinct molecular mechanisms might have been pre-programmed at an early stage in different subtypes of the disease. Our results provide further evidence that these breast tumor subtypes represent biologically distinct disease entities and may require different therapeutic strategies. Finally, validated by multiple gene expression platforms, including quantitative PCR, the set of 54 predictor genes identified in this study may define potential prognostic molecular markers for breast cancer.

Long versus short oligonucleotide microarrays for the study of gene expression in nonhuman primates

The high degree of sequence similarity between human and nonhuman primate (NHP) genomic DNA suggests that human genome sequence-based DNA microarrays may be used effectively to study gene expression in NHP disease models. In the present study, two distinct commercially available human genome microarray platforms, the Affymetrix HG U133A GeneChip ® System utilizing Human Genome U133A GeneChips ® and the Applied Biosystems Expression Array System utilizing the Human Genome Survey Microarray, were used to identify and characterize gene expression changes in the anterior cerebellum of a macaque monkey model of human alcoholism. The Affymetrix microarray consists of eleven short oligonucleotide probe sets for each gene while the Applied Biosystems Microarray uses a single long oligonucleotide per gene. Cross-mapping of probes revealed a total of 11,542 genes that are represented on both microarray platforms. Absolute measures of gene expression (“present calls”) from the cerebellum RNA samples were 65–70% (Applied Biosystems Expression Array System) and 27–30% (AffymetrixGeneChip ® System) among these common gene targets. Analysis of variance (ANOVA; p < 0.05; >1.2 fold change; detected on at least 50% of the arrays) indicated 932 and 515 differentially expressed genes for the Applied Biosystems and Affymetrix microarrays, respectively. Significance analysis of microarrays (SAM) identified 255 significant genes at 5% false discovery rate (FDR) for the Applied Biosystems data set and five significant genes at 60% FDR (minimum FDR) for the Affymetrix data set. TaqMan ® assay-based real-time PCR validation of a number of differentially-expressed genes yielded results that agreed well with the array data in the majority of comparisons. This study demonstrates that human sequence-based DNA arrays can be used effectively to detect differential gene expression in an NHP disease model and provides evidence that the use of this long oligonucleotide-based microarray platform may be more suitable for cross-species gene expression studies than a short oligonucleotide-based system.