Generated Gene Expression Profiles Research Articles

Predicting the efficacy of anthracyclines in breast cancer (BC) patients: Results of the neoadjuvant TOP trial

523 Background: Anthracycline-based regimens are among the most active and used chemotherapies in BC. Here, we report the results of the prospective TOP trial ( NCT00162812 ) which was specifically designed to identify markers of response/resistance to preoperative epirubicin (100 mg/m2 q2 or q3wks) in estrogen receptor-negative (ER-) BC patients. Methods: Using pre-epirubicin biopsies, we analyzed TOP2A by fluorescence in situ hybridization (Abbott triple probe) and by immunohistochemistry (KiS1) and generated gene expression profiles (HG133Plus2.0). We used publicly available gene expression data from the BIG1–00, trial (EORTC-10094), which compared in a preoperative setting an anthracycline-based (A) to a taxane-based (T) regimen, to validate some of our results. Results: 149 patients were included in the TOP trial and 14.5% had a complete pathological response (pCR). TOP2A amplification (11% of the cases) was exclusively observed in HER-2 amplified cases (33% of the cases), and was highly predictive of pCR (k = 0.35, p = 0.0002 - half of the patients with TOP2A amplification experienced pCR). Neither the protein levels nor the mRNA levels were predictive of pCR. Chromosome17 (Ch17) polysomy was observed in 68% of the samples and was not associated with pCR. TOP2A gene expression module (made of genes located closely to TOP2A), was predictive of response both in the HER-2+ patients of the TOP trial and in the ER-/HER-2+ of the BIG1–00 patients treated with A but not with T [Area Under the Curve (AUC): 0.86 (p = 0.00001) in TOP trial and 0.81 (p = 0.0005) in A-arm BIG1–00 trial]. Stroma expression module defined previously was associated with pCR in both trials (TOP and in the ER- BIG1–00 patients treated with A but not with T, especially in HER-2- patients [AUC: 0.31 (p = 0.01) in TOP trial and 0.33 (p = 0.02) in A-arm/BIG1–00 trial]. Conclusions: This trial, which is the first to prospectively evaluate the predictive value of TOP2A, supports TOP2A gene amplification as predictive marker of response. Ch17 polysomy, observed in ∼2/3 of these ER- patients, was not predictive of response. Finally, our results suggest that gene expression modules also predict relative response/resistance to anthracyclines, both in HER-2+ and HER-2- patients. No significant financial relationships to disclose.

The unique molecular signatures of nodular and superficial spreading melanoma

9047 Background: Primary nodular melanoma (NM) patients have a relatively poor prognosis compared to superficial spreading melanoma (SSM) patients. The disparity is generally attributed solely to NM's advanced thickness at presentation. In this study we attempted to define molecular signatures of NM and SSM that may explain their clinical differences. Methods: We performed an in silico gene expression analysis of 2 public data sets consisting of 36NM and 54 SSM primary melanoma tissues (CCR 2007;13 and JNCI 2006;98). We then utilized DNA microarray to generate gene expression profiles of a panel of 22 melanoma cell lines (2SSM, 4 NM, 12 met, 4 melanocytes). Differentially expressed genes and over-represented pathways in NM and SSM were identified based on a pooled analysis of the 3 data sets. We then used SNP array to define genomic alterations unique to NM and SSM but not altered in normal melanocytes. Finally, we correlated SNP array with gene expression. Results: Genes significantly overexpressed (p<0.05) in NM showed over-representation of pathways related to MAPK signaling (p=0.05) and cytoskeleton organization (p=0.02), while SSM showed over-representation of cell communication (p=0.05) and primary metabolic processes (p=0.002). Notable correlations between gene expression and copy number alteration in NM include increased copy number/overexpression of SOX5 (transcription factor related to embryonic development and cell fate) and the downregulation/deletion of ST14 (suppression of tumorigenicity 14). SSM demonstrated concordance of increased copy number/overexpression of EZR (cell adhesion protein implicated in human cancer) as well as PALLD (a protein related to motility, adhesion, and extracellular matrix interactions). Notable SSM genes showing correlation between downregulation/deletion include BNIP3 (a pro-apoptotic protein) and MTAP (often co- deleted with tumor suppressor p16). Conclusions: Simultaneous integration of gene expression with SNP array revealed molecular signatures characteristic of NM and SSM. These results suggest that NM and SSM are distinct biologic entities and that molecularly targeted adjuvant therapy may be more effective if tailored to the molecular signatures of melanoma subtypes. Validation is necessary to draw further conclusions. No significant financial relationships to disclose.

NA-Seq: A Discovery Tool for the Analysis of Chromatin Structure and Dynamics during Differentiation

It is well established that epigenetic modulation of genome accessibility in chromatin occurs during biological processes. Here we describe a method based on restriction enzymes and next-generation sequencing for identifying accessible DNA elements using a small amount of starting material, and use it to examine myeloid differentiation of primary human CD34+ cells. The accessibility of several classes of cis-regulatory elements was a predictive marker of in vivo DNA binding by transcription factors, and was associated with distinct patterns of histone posttranslational modifications. We also mapped large chromosomal domains with differential accessibility in progenitors and maturing cells. Accessibility became restricted during differentiation, correlating with a decreased number of expressed genes and loss of regulatory potential. Our data suggest that a permissive chromatin structure in multipotent cells is progressively and selectively closed during differentiation, and illustrate the use of our method for the identification of functional cis-regulatory elements.

Definition, conservation and epigenetics of housekeeping and tissue-enriched genes

BackgroundHousekeeping genes (HKG) are constitutively expressed in all tissues while tissue-enriched genes (TEG) are expressed at a much higher level in a single tissue type than in others. HKGs serve as valuable experimental controls in gene and protein expression experiments, while TEGs tend to represent distinct physiological processes and are frequently candidates for biomarkers or drug targets. The genomic features of these two groups of genes expressed in opposing patterns may shed light on the mechanisms by which cells maintain basic and tissue-specific functions.ResultsHere, we generate gene expression profiles of 42 normal human tissues on custom high-density microarrays to systematically identify 1,522 HKGs and 975 TEGs and compile a small subset of 20 housekeeping genes which are highly expressed in all tissues with lower variance than many commonly used HKGs. Cross-species comparison shows that both the functions and expression patterns of HKGs are conserved. TEGs are enriched with respect to both segmental duplication and copy number variation, while no such enrichment is observed for HKGs, suggesting the high expression of HKGs are not due to high copy numbers. Analysis of genomic and epigenetic features of HKGs and TEGs reveals that the high expression of HKGs across different tissues is associated with decreased nucleosome occupancy at the transcription start site as indicated by enhanced DNase hypersensitivity. Additionally, we systematically and quantitatively demonstrated that the CpG islands' enrichment in HKGs transcription start sites (TSS) and their depletion in TEGs TSS. Histone methylation patterns differ significantly between HKGs and TEGs, suggesting that methylation contributes to the differential expression patterns as well.ConclusionWe have compiled a set of high quality HKGs that should provide higher and more consistent expression when used as references in laboratory experiments than currently used HKGs. The comparison of genomic features between HKGs and TEGs shows that HKGs are more conserved than TEGs in terms of functions, expression pattern and polymorphisms. In addition, our results identify chromatin structure and epigenetic features of HKGs and TEGs that are likely to play an important role in regulating their strikingly different expression patterns.

Identification of brain- and bone-specific breast cancer metastasis genes

In breast cancer, metastases are relatively widely distributed, with the most common sites being bone, regional lymph nodes, lung, liver, and brain. The detailed mechanism of organ-specific metastasis is poorly understood. In this study, we initiated a search for genes that are implicated in brain or bone metastasis of primary human breast cancer. We generated gene expression profiles of 18 brain and eight bone metastases derived from primary breast tumors. We identified 73 genes differentially expressed between brain and bone metastases. Visualization of the differential gene expression profiles by correspondence and cluster analyses shows that the metastases clearly separate into two distinct groups as an exact reflection of their site of metastasis. Moreover, the analysis of this gene set in primary breast tumors relapsing to either bone or brain allowed accurate categorization of the tumors according to their metastatic site. The identified genes may prove to be excellent markers to predict the site of metastasis in breast cancer patients and could lead to tailor-made therapy to an individual patient.

Identification of Gene Expression Signatures Accurately Predicting Cytogenetic Subtypes in Pediatric Acute Myeloid Leukemia.

Pediatric acute myeloid leukemia (AML) is a heterogeneous disease, which is classified according to the WHO classification, based on morphology, immunophenotyping and non-random genetic aberrations. AML is hypothesized to arise from two different types of genetic aberrations, i.e. type-I (proliferation enhancing) mutations and type-II (differentiation impairing) mutations. To detect genetic aberrations multiple techniques such as conventional karyotyping, FISH and RT-PCR are being used. In addition to conventional karyotyping, the latter two techniques revealed a higher frequency of aberrations. Still, failures or false negative results should be taken into account. Recent studies have focused on the potential of gene expression profiling (GEP) to classify acute leukemias. To study the clinical value of classification by GEP, we first used a double-loop cross validation (CV) to avoid over-fitting of GEP data and, subsequently, addressed whether the identified GEP was suitable to classify pediatric AML cases in a second independent group of cases. Affymetrix Human Genome U133 plus 2.0 microarrays were used to generate gene expression profiles of 257 children with AML, with high blast counts, if necessary, after enrichment (~80% or more) and good quality RNA. Probe set intensities were normalized using the variance-stabilizing normalization (VSN) implemented in R (version 2.2.0). The patient group was divided into a test cohort (n=170) and an independent validation cohort (n=87). The test cohort was used to construct the classifier using two levels of CV:the minimum number of predictive genes was estimated using a 10-fold CV on random subsets of about 113 (~2/3 of total) patients;the accuracy of the obtained classifier is estimated on the remaining 57 (~1/3) patients. Candidate genes to represent the GEP in the classifier were those genes that discriminated AML subtypes according to an empirical Bayes linear regression model (Bioconductor package: Limma).To construct a reliable classifier it was sufficient to use 75 probe sets, representing the top 15 discriminating probe sets for MLL-gene rearranged AML, t(8;21), inv(16), t(15;17) and t(7;12). These subtypes represented ~50% of the included patients. The remaining patients either had normal cytogenetics, random aberrations or no data available (cytogenetic failure). Due to the heterogeneity of these remaining groups discriminative probe sets were not found. This classifier could reliably predict the 5 subtypes with a median accuracy of 93%. Validation of the classifier on the independent cohort confirmed that the sensitivity and accuracy was more than 99%. No gene expression signatures could be found for the molecular aberrations NPM1, CEBPa, MLL-PTD, FLT3, C-KIT, RAS or PTPN1, possibly due to the small number of cases. However, specific gene expression signatures were found for FLT3-ITD within the subset of cases with t(15;17) or normal cytogenetics. Importantly, a high expression of HOXB-cluster related genes was found in cases with FLT3-ITD and normal cytogenetics. In conclusion, GEP can correctly predict several important cytogenetic subtypes of pediatric AML, including cases that are currently classified using different cytogenetic techniques and cases with failed cytogenetic analysis. Prospective studies are needed to validate the use of GEP in the classification of pediatric AML, especially to provide information on its utility in clinical practice. Increasing numbers in rare subtypes may result in the discovery of genes discriminative for them, and may foster GEP as a new diagnostic tool.

O.258 3D control of resection margins in oral cancer based on PET/CT image-fusion

Background & Aims: Neoadjuvant chemoradiotherapy has become a standard treatment of locally advanced rectal carcinomas, even though the responsiveness varies from complete response to resistance. The aim of the study was to evaluate the capacity of gene expression signatures to identify responders and nonresponders pretherapeutically. Methods: By using microarray technology we generated gene expression profiles of 43 biopsy specimens of locally advanced rectal carcinomas. The transcription profile then was compared with histopathologic response and used to identify a set of genes discriminating responders from nonresponders. Results: We identified a gene expression signature of 42 genes, mostly encoding proteins that either play a role in the nucleus, such as the transcription factor ETS2, or are associated with transport function, such as the solute carrier SLC35E1, or the regulation of apoptosis, such as caspase-1. In leave-one-out cross-validation the correct classification of a responder was 71%, the specificity of the analysis for a correct classification of a nonresponder was 86%. By applying an additional statistical method of 200 successive splittings into training and test data sets we generated an individual prediction accuracy measure for each predicted response. Conclusions: Our study shows that pretherapeutic prediction of response of rectal carcinomas to neoadjuvant chemoradiotherapy is feasible, and may represent a new valuable and practical tool of therapeutic stratification.

Genomic Deletions Correlate with Underexpression of Novel Candidate Genes at Six Loci in Pediatric Pilocytic Astrocytoma

The molecular pathogenesis of pediatric pilocytic astrocytoma (PA) is not well defined. Previous cytogenetic and molecular studies have not identified nonrandom genetic aberrations. To correlate differential gene expression and genomic copy number aberrations (CNAs) in PA, we have used Affymetrix GeneChip HG_U133A to generate gene expression profiles of 19 pediatric patients and the SpectralChip 2600 to investigate CNAs in 11 of these tumors. Hierarchical clustering according to expression profile similarity grouped tumors and controls separately. We identified 1844 genes that showed significant differential expression between tumor and normal controls, with a large number clearly influencing phosphatidylinositol and mitogen-activated protein kinase signaling in PA. Most CNAs identified in this study were single-clone alterations. However, a small region of loss involving up to seven adjacent clones at 7q11.23 was observed in seven tumors and correlated with the underexpression of BCL7B. Loss of four individual clones was also associated with reduced gene expression including SH3GL2 at 9p21.2-p23, BCL7A (which shares 90% sequence homology with BCL7B) at 12q24.33, DRD1IP at 10q26.3, and TUBG2 and CNTNAP1 at 17q21.31. Moreover, the down-regulation of FOXG1B at 14q12 correlated with loss within the gene promoter region in most tumors. This is the first study to correlate differential gene expression with CNAs in PA.

Tracing studies in rat provide sensitive gene expression data for neurons innervating the bladder and urethra

The aim of the study was to explore novel tissue based techniques to generate gene expression profiles of both afferent (sensory) and efferent (motor) neurons that specifically innervate the rat bladder and urethra.Retrograde neuronal tracing studies in combination with laser capture microdissection (LCM) and microarray analysis were used to generate gene expression profiles of both afferent and efferent neurons that specifically innervate the rat bladder and urethra. A complete rat study (n=12) was performed in which fluorescently labelled cholera toxin B was injected into the bladder and urethra. Traced neurons were isolated by LCM, RNA was extracted, amplified and analysed by microarray.Extensive analysis of all the data derived from this study give very sensitive and powerful gene expression data from only 50–100 microdissected neurons. Expression of sensory neuron specific markers could be confirmed in dorsal root ganglion neurons whereas expression of genes involved in motor function of the lower urinary tract could be confirmed in efferent neurons e.g. Onuf's Nucleus.The resulting gene expression profile will increase our understanding of signalling pathways involved in the control of the lower urinary tract. Data derived from this study will not only be relevant for safety and efficacy but will also enable us to identify targets for the treatment of urinary frequency and urge incontinence (UFUI).

Identification of immunohistochemical biomarkers for papillary thyroid carcinoma using gene expression profiling

We report the successful validation of a combined gene expression profiling and tissue microarray approach to papillary thyroid carcinoma (PTC) biomarker identification. Our ultimate goal is the identification of protein biomarkers that can be effectively used in immunocytochemical assays applied to thyroid fine needle aspiration biopsy (FNAB) samples. To that end, we designed our approach to prioritize molecules that were minimally expressed in normal thyroid and highly expressed in PTC. We first generated gene expression profiles from 11 normal thyroid tissue samples and 9 samples of classic PTC. The results were segregated to rank most highly those molecules not expressed in normal thyroid and up-regulated at least 6-fold in PTC. From this list, we chose 2 molecules (P-cadherin and Bax) for immunohistochemical analysis for which commercial antibodies were available. These were compared with 2 other molecules that have been previously studied in thyroid cancer (cytokeratin-19 and galectin-3). For immunohistochemistry, a tissue microarray was generated that contained the following tissues: classic PTC (n = 20), follicular variant of PTC (n = 9), normal thyroid (n = 19), Hashimoto thyroiditis (n = 11), follicular adenoma (n = 15), and follicular carcinoma (n = 14). Immunohistochemical staining was scored and compared with the gene expression profiling. As anticipated, cytokeratin-19 and galectin-3 were highly expressed in PTC and less expressed in other tissues. Bax and P-cadherin were also expressed in PTC, but to a lower level than cytokeratin-19 and galectin-3; however, Bax and P-cadherin demonstrated virtually no staining of normal thyroid, unlike cytokeratin-19 and galectin-3. These results validate our approach for PTC biomarker discovery and identify several candidate biomarkers for further development.

Microarray-Based Prediction of Tumor Response to Neoadjuvant Radiochemotherapy of Patients With Locally Advanced Rectal Cancer

Neoadjuvant chemoradiotherapy has become a standard treatment of locally advanced rectal carcinomas, even though the responsiveness varies from complete response to resistance. The aim of the study was to evaluate the capacity of gene expression signatures to identify responders and nonresponders pretherapeutically. By using microarray technology we generated gene expression profiles of 43 biopsy specimens of locally advanced rectal carcinomas. The transcription profile then was compared with histopathologic response and used to identify a set of genes discriminating responders from nonresponders. We identified a gene expression signature of 42 genes, mostly encoding proteins that either play a role in the nucleus, such as the transcription factor ETS2, or are associated with transport function, such as the solute carrier SLC35E1, or the regulation of apoptosis, such as caspase-1. In leave-one-out cross-validation the correct classification of a responder was 71%, the specificity of the analysis for a correct classification of a nonresponder was 86%. By applying an additional statistical method of 200 successive splittings into training and test data sets we generated an individual prediction accuracy measure for each predicted response. Our study shows that pretherapeutic prediction of response of rectal carcinomas to neoadjuvant chemoradiotherapy is feasible, and may represent a new valuable and practical tool of therapeutic stratification.

Susceptibility of chimeric mice with livers repopulated by serially subcultured human hepatocytes to hepatitis B virus

We previously identified a small population of replicative hepatocytes in long-term cultures of human adult parenchymal hepatocytes (PHs) at a frequency of 0.01%-0.09%. These hepatocytes were able to grow continuously through serial subcultures as colony-forming parenchymal hepatocytes (CFPHs). In the present study, we generated gene expression profiles for cultured CFPHs and found that they expressed cytokeratin 19, CD90 (Thy-1), and CD44, but not mature hepatocyte markers such as tryptophan-2,3-dioxygenase (TO) and glucose-6-phosphatase (G6P), confirming that these cells are hepatic progenitor-like cells. The cultured CFPHs were resistant to infection with human hepatitis B virus (HBV). To examine the growth and differentiation capacity of the cells in vivo, serially subcultured CFPHs were transplanted into the progeny of a cross between albumin promoter/enhancer-driven urokinase plasminogen activator-transgenic mice and severe combined immunodeficient (SCID) mice. The cells were engrafted into the liver and were able to grow for at least 10 weeks, ultimately reaching a maximum occupancy rate of 27%. The CFPHs in the host liver expressed differentiation markers such as TO, G6P, and cytochrome P450 subtypes and could be infected with HBV. CFPH-chimeric mice with a relatively high replacement rate exhibited viremia and had high serum levels of hepatitis B surface antigen. Serially subcultured human hepatic progenitor-like cells from postnatal livers successfully repopulated injured livers and exhibited several phenotypes of mature hepatocytes, including susceptibility to HBV. In vitro-expanded CFPHs can be used to characterize the differentiation state of human hepatic progenitor-like cells.

Dickkopf-1 is down-regulated by MYCN and inhibits neuroblastoma cell proliferation

Neuroblastomas are tumors of the developing peripheral sympathetic nervous system, which originates from the neural crest. Twenty percent of neuroblastomas show amplification of the MYCN oncogene, which correlates with poor prognosis. The MYCN transcription factor can activate and repress gene expression. To broaden our insight in the spectrum of genes down-regulated by MYCN, we generated gene expression profiles of the neuroblastoma cell lines SHEP-21N and SKNAS-NmycER, in which MYCN activity can be regulated. In this study, we show that MYCN suppresses the expression of Dickkopf-1 (DKK1) in both cell lines. DKK1 is a potent inhibitor of the wnt/β-catenin signalling cascade, which is known to function in neural crest cell migration. We generated a DKK1 inducible cell line, IMR32-DKK1, which showed impaired proliferation upon DKK1 expression. Surprisingly, DKK1 expression did not inhibit the canonical wnt/β-catenin signalling, suggesting a role of DKK1 in an alternative route of the wnt pathway. Gene expression profiling of two IMR32-DKK1 clones showed that only a few genes, amongst which SYNPO2, were up-regulated by DKK1. SYNPO2 encodes an actin-binding protein and was previously found to inhibit proliferation and invasiveness of prostate cancer cells. These results suggest that MYCN might stimulate cell proliferation by inhibiting the expression of DKK1. DKK1 might exert part of its growth suppressive effect by induction of SYNPO2 expression.

Laser capture microdissection and cDNA microarrays used to generate gene expression profiles of the rapidly expanding fibre initial cells on the surface of cotton ovules

Cotton (Gossypium hirsutum L.) fibre initial cells undergo a rapid cellular re-programming around anthesis to form the long cellulose fibres prized for textile manufacture. On the day of anthesis the cotton fibre initial cells balloon out from the ovule surface and so are clearly distinguished from adjacent epidermal pavement cells. To enhance our understanding of the molecular processes that determine which cells become fibres and why adjacent epidermal cells remain in a different developmental state we studied the expression profiles of the two respective cell types. Using laser-capture microdissection, coupled with an in vitro RNA amplification system, we used cDNA microarray slides to profile the gene expression in expanding fibre initials compared to the non-expanding epidermal cells at an early stage just after the fibre initials are discernable. Except for a few regulatory genes, the genes that are up-regulated in the cotton fibre initials relative to epidermal cells predominantly encode proteins involved in generating the components for the extra cell membrane and primary cell wall needed for the rapid cell expansion of the initials. This includes synthesis of enzymes and cell wall proteins, carbohydrates, and lipids. An analysis of single channel fluorescence levels confirmed that these classes of genes were also the most highly expressed genes in fibre initials. Genes involved in DNA metabolism were also well represented in the expanding fibre cell, consistent with the limited endoreduplication we previously reported to occur in fibre initial cells.

Review of:Estrogen receptor β expression is associated with tamoxifen response in ERα-negative breast carcinoma

Citation of original article:S. K. Gruvberger-Saal, P.-O. Bendahl, L. H. Saal, M. Laakso, C. Hegardt, P. Edeén, C. Peterson, P. Malmström, J. Isola, A. Borg, M. Fernoö.Clinical Cancer Research2007;13: 1987–1994.Abstract of the original article:PurposeEndocrine therapies, such as tamoxifen, are commonly given to most patients with estrogen receptor (ERα)-positive breast carcinoma but are not indicated for persons with ERα-negative cancer. The factors responsible for response to tamoxifen in 5% to 10% of patients with ERα-negative tumors are not clear. The aim of the present study was to elucidate the biology and prognostic role of the second ER, ERβ, in patients treated with adjuvant tamoxifen.Experimental designWe investigated ERβ by immunohistochemistry in 353 stage II primary breast tumors from patients treated with 2 years adjuvant tamoxifen, and generated gene expression profiles for a representative subset of 88 tumors.ResultsERβ was associated with increased survival (distant disease-free survival,P= 0.01; overall survival,P= 0.22), and in particular within ERα-negative patients (P= 0.003;P= 0.04), but not in the ERα-positive subgroup (P= 0.49;P= 0.88). Lack of ERβ conferred early relapse (hazards ratio, 14; 95% confidence interval, 1.8–106;P= 0.01) within the ERα-negative subgroup even after adjustment for other markers. ERα was an independent marker only within the ERβ-negative tumors (hazards ratio, 0.44; 95% confidence interval, 0.21–0.89;P= 0.02). An ERβ gene expression profile was identified and was markedly different from the ERα signature.ConclusionExpression of ERβ is an independent marker for favorable prognosis after adjuvant tamoxifen treatment in ERα-negative breast cancer patients and involves a gene expression program distinct from ERα. These results may be highly clinically significant, because in the United States alone, approximately 10 000 women are diagnosed annually with ERα-negative/ERβ-positive breast carcinoma and may benefit from adjuvant tamoxifen.

Synergistic antitumor efficacy and altered gene expression signature in breast cancer cells treated with immunotoxins and cyclosporin A

13505 Background: Immunotoxins (ITs) has shown limited clinical success related to liver toxicity and development of anti-IT antibodies. To delay the immune response we tested combinations of ITs and the Cyclosporin A (CsA). we have shown that one IT, currently in a phase I/II clinical trial, acts by inducing apoptosis and protein synthesis inhibition, but gene expression analysis of IT treated cells has never been reported. Hence, we also studied changes in gene expression induced by ITs alone and the effects of adding (CsA) on both treatment efficacy and gene expression signature. Methods: Human MA-11 breast cancer cells were treated in vitro with antiEGFR- and antiEPCAM-based ITs alone and in combinations with CsA. Therapeutic efficay was assessed by MTS cell viability assay. Total RNA from untreated and treated cells was isolated and CodeLink Uniset Human 20 k Oligo Bioarray (GE Healthcare, Amersham Biosciences, NJ), containing approximately 20,289 gene probes, was used to generate gene expression profiles. Gene expression analysis was carried out using GeneSpring software version 7.2 using One-way ANOVA with p<0.05. Comparisons of gene list across different groups were performed using Venn Diagrams. Results: Combination therapy produced remarkable synergistic effects in MA-11 cells in vitro and in metastasis models in vivo. Moreover, in conventional rats receiving repeated injections of ITs and CsA the formation of anti-IT antibodies was virtually abrogated. Changes in gene expression profiles induced by the ITs alone and in combination with CsA were evaluated to elucidate the underlying molecular mechanisms for the synergistic effects. The ITs each induced specific changes in expression of some apoptosis-related genes but also fogenes in pathways unrelated to apoptosis and protein synthesis. The addition of CsA induced up- or down-regulation of a number of interesting non-immune-associated genes Conclusions: Important shortcomings for successful clinical use of ITs may be overcome by combination therapy with CsA. The possibility for further improvement is provided by results of gene profiling studies identifying therapy-induced genes belonging to different cell signaling pathways. No significant financial relationships to disclose.

Neural crest and mesoderm lineage-dependent gene expression in orofacial development

The present study utilizes a combination of genetic labeling/selective isolation of pluripotent embryonic progenitor cells, and oligonucleotide-based microarray technology, to delineate and compare the “molecular fingerprint” of two mesenchymal cell populations from distinct lineages in the developing embryonic orofacial region. The first branchial arches—bi-lateral tissue primordia that flank the primitive oral cavity—are populated by pluripotent mesenchymal cells from two different lineages: neural crest (neuroectoderm)- and mesoderm-derived mesenchymal cells. These cells give rise to all of the connective tissue elements (bone, cartilage, smooth and skeletal muscle, dentin) of the orofacial region (maxillary and mandibular portion), as well as neurons and glia associated with the cranial ganglia, among other tissues. In the present study, neural crest- and mesoderm-derived mesenchymal cells were selectively isolated from the first branchial arch of gestational day 9.5 mouse embryos using laser capture microdissection (LCM). The two different embryonic cell lineages were distinguished through utilization of a novel two component transgenic mouse model (Wnt1Cre/ZEG) in which the neural crest cells and their derivatives are indelibly marked (i.e., expressing enhanced green fluorescent protein, EGFP) throughout the pre- and post-natal lifespan of the organism. EGFP-labeled neural crest-derived, and non-fluorescent mesoderm-derived mesenchymal cells from the first branchial arch were visualized in frozen tissue sections from gestational day 9.5 mouse embryos and independently isolated by LCM under epifluorescence optics. RNA was extracted from the two populations of LCM-procured cells, and amplified by double-stranded cDNA synthesis and in vitro transcription. Gene expression profiles of the two progenitor cell populations were generated via hybridization of the cell-type specific cRNA samples to oligo-based GeneChip microarrays. Comparison of gene expression profiles of neural crest- and mesoderm-derived mesenchymal cells from the first branchial arch revealed over 140 genes that exhibited statistically significant differential levels of expression. The gene products of many of these differentially expressed genes have previously been linked to the development of mesoderm- or neural crest-derived tissues in the embryo. Interestingly, however, hitherto uncharacterized coding sequences with highly significant differences in expression between the two embryonic progenitor cell types were also identified. These lineage-dependent mesenchymal cell molecular fingerprints offer the opportunity to elucidate additional mechanisms governing cellular growth, differentiation, and morphogenesis of the embryonic orofacial region. The chemokine stromal cell-derived factor 1, (SDF-1), was found to exhibit greater expression in mesoderm-derived mesenchyme in the branchial arch when compared with neurectoderm, suggesting a possible chemotactic role for SDF-1 in guiding the migratory neural crest cells to their destination. The novel combination of genetic labeling of the neural crest cell population by EGFP coupled with isolation of cells by LCM for gene expression analysis has enabled, for the first time, the generation of gene expression profiles of distinct embryonic cell lineages.

Estrogen Receptor β Expression Is Associated with Tamoxifen Response in ERα-Negative Breast Carcinoma

Endocrine therapies, such as tamoxifen, are commonly given to most patients with estrogen receptor (ERalpha)-positive breast carcinoma but are not indicated for persons with ERalpha-negative cancer. The factors responsible for response to tamoxifen in 5% to 10% of patients with ERalpha-negative tumors are not clear. The aim of the present study was to elucidate the biology and prognostic role of the second ER, ERbeta, in patients treated with adjuvant tamoxifen. We investigated ERbeta by immunohistochemistry in 353 stage II primary breast tumors from patients treated with 2 years adjuvant tamoxifen, and generated gene expression profiles for a representative subset of 88 tumors. ERbeta was associated with increased survival (distant disease-free survival, P = 0.01; overall survival, P = 0.22), and in particular within ERalpha-negative patients (P = 0.003; P = 0.04), but not in the ERalpha-positive subgroup (P = 0.49; P = 0.88). Lack of ERbeta conferred early relapse (hazard ratio, 14; 95% confidence interval, 1.8-106; P = 0.01) within the ERalpha-negative subgroup even after adjustment for other markers. ERalpha was an independent marker only within the ERbeta-negative tumors (hazard ratio, 0.44; 95% confidence interval, 0.21-0.89; P = 0.02). An ERbeta gene expression profile was identified and was markedly different from the ERalpha signature. Expression of ERbeta is an independent marker for favorable prognosis after adjuvant tamoxifen treatment in ERalpha-negative breast cancer patients and involves a gene expression program distinct from ERalpha. These results may be highly clinically significant, because in the United States alone, approximately 10,000 women are diagnosed annually with ERalpha-negative/ERbeta-positive breast carcinoma and may benefit from adjuvant tamoxifen.

Gene expression signatures of morphologically normal breast tissue identify basal-like tumors

IntroductionThe role of the cellular microenvironment in breast tumorigenesis has become an important research area. However, little is known about gene expression in histologically normal tissue adjacent to breast tumor, if this is influenced by the tumor, and how this compares with non-tumor-bearing breast tissue.MethodsTo address this, we have generated gene expression profiles of morphologically normal epithelial and stromal tissue, isolated using laser capture microdissection, from patients with breast cancer or undergoing breast reduction mammoplasty (n = 44).ResultsBased on this data, we determined that morphologically normal epithelium and stroma exhibited distinct expression profiles, but molecular signatures that distinguished breast reduction tissue from tumor-adjacent normal tissue were absent. Stroma isolated from morphologically normal ducts adjacent to tumor tissue contained two distinct expression profiles that correlated with stromal cellularity, and shared similarities with soft tissue tumors with favorable outcome. Adjacent normal epithelium and stroma from breast cancer patients showed no significant association between expression profiles and standard clinical characteristics, but did cluster ER/PR/HER2-negative breast cancers with basal-like subtype expression profiles with poor prognosis.ConclusionOur data reveal that morphologically normal tissue adjacent to breast carcinomas has not undergone significant gene expression changes when compared to breast reduction tissue, and provide an important gene expression dataset for comparative studies of tumor expression profiles.

Gene Expression Profiles in Rat Liver Slices Exposed to Hepatocarcinogenic Enzyme Inducers, Peroxisome Proliferators, and 17α-Ethinylestradiol

Transcription profiling is used as an in vivo method for predicting the mode-of-action class of nongenotoxic carcinogens. To set up a reliable in vitro short-term test system DNA microarray technology was combined with rat liver slices. Seven compounds known to act as tumor promoters were selected, which included the enzyme inducers phenobarbital, alpha-hexachlorocyclohexane, and cyproterone acetate; the peroxisome proliferators WY-14,643, dehydroepiandrosterone, and ciprofibrate; and the hormone 17alpha-ethinylestradiol. Rat liver slices were exposed to various concentrations of the compounds for 24 h. Toxicology-focused TOXaminer DNA microarrays containing approximately 1500 genes were used for generating gene expression profiles for each of the test compound. Hierarchical cluster analysis revealed that (i) gene expression profiles generated in rat liver slices in vitro were specific allowing classification of compounds with similar mode of action and (ii) expression profiles of rat liver slices exposed in vitro correlate with those induced after in vivo treatment (reported previously). Enzyme inducers and peroxisome proliferators formed two separate clusters, confirming that they act through different mechanisms. Expression profiles of the hormone 17alpha-ethinylestradiol were not similar to any of the other compounds. In conclusion, gene expression profiles induced by compounds that act via similar mechanisms showed common effects on transcription upon treatment in vivo and in rat liver slices in vitro.