Variance-Preserving Estimation of Intensity Values Obtained From Omics Experiments.

Faced with the lack of reliability and reproducibility in omics studies, more careful and robust methods are needed to overcome the existing challenges in the multi-omics analysis. In conventional omics data analysis, signal intensity values (denoted by M and values) are estimated neglecting pixel-level uncertainties, which may reflect noise and systematic artifacts. For example, intensity values from two-color microarray data are estimated by taking the mean or median of the pixel intensities within the spot and then subjected to a within-slide normalization by LOWESS. Thus, focusing on estimation and normalization of gene expression profiles, we propose a spot quantification method that takes into account pixel-level variability. Also, to preserve relevant variation that may be removed in LOWESS normalization with poorly chosen parameters, we propose a parameter selection method that is parsimonious and considers intrinsic characteristics of microarray data, such as heteroskedasticity. The usefulness of the proposed methods is illustrated by an application to real intestinal metaplasia data. Compared with the conventional approaches, the analysis is more robust and conservative, identifying fewer but more reliable differentially expressed genes. Also, the variability preservation allowed the identification of new differentially expressed genes. Using the proposed approach, we have identified differentially expressed genes involved in pathways in cancer and confirmed some molecular markers already reported in the literature.

Expression profile of malignant and nonmalignant lesions of esophagus and stomach: differential activity of functional modules related to inflammation and lipid metabolism.

Adenocarcinomas of stomach and esophagus are frequently associated with preceding inflammatory alterations of the normal mucosa. Whereas intestinal metaplasia of the gastric mucosa is associated with higher risk of malignization, Barrett's disease is a risk factor for adenocarcinoma of the esophagus. Barrett's disease is characterized by the substitution of the squamous mucosa of the esophagus by a columnar tissue classified histopathologically as intestinal metaplasia. Using cDNA microarrays, we determined the expression profile of normal gastric and esophageal mucosa as well as intestinal metaplasia and adenocarcinomas from both organs. Data were explored to define functional alterations related to the transformation from squamous to columnar epithelium and the malignant transformation from intestinal metaplasia to adenocarcinomas. Based on their expression profile, adenocarcinomas of the esophagus showed stronger correlation with intestinal metaplasia of the stomach than with Barrett's mucosa. Second, we identified two functional modules, lipid metabolism and cytokine, as being altered with higher statistical significance. Whereas the lipid metabolism module is active in samples representing intestinal metaplasia and inactive in adenocarcinomas, the cytokine module is inactive in samples representing normal esophagus and esophagitis. Using the concept of relevance networks, we determined the changes in linear correlation of genes pertaining to these two functional modules. Exploitation of the data presented herein will help in the precise molecular characterization of adenocarcinoma from the distal esophagus, avoiding the topographical and descriptive classification that is currently adopted, and help with the proper management of patients with Barrett's disease.

Molecular classifiers for gastric cancer and nonmalignant diseases of the gastric mucosa.

High incidence of gastric cancer-related death is mainly due to diagnosis at an advanced stage in addition to the lack of adequate neoadjuvant therapy. Hence, new tools aimed at early diagnosis would have a positive impact in the outcome of the disease. Using cDNA arrays having 376 genes either identified previously as altered in gastric tumors or known to be altered in human cancer, we determined expression signature of 99 tissue fragments representing normal gastric mucosa, gastritis, intestinal metaplasia, and adenocarcinomas. We first validated the array by identifying molecular markers that are associated with intestinal metaplasia, considered as a transition stage of gastric adenocarcinomas of the intestinal type as well as markers that are associated with diffuse type of gastric adenocarcinomas. Next, we applied Fisher's linear discriminant analysis in an exhaustive search of trios of genes that could be used to build classifiers for class distinction. Many classifiers could distinguish between normal and tumor samples, whereas, for the distinction of gastritis from tumor and for metaplasia from tumor, fewer classifiers were identified. Statistical validations showed that trios that discriminate between normal and tumor samples are powerful classifiers to distinguish between tumor and nontumor samples. More relevant, it was possible to identify samples of intestinal metaplasia that have expression signature resembling that of an adenocarcinoma and can now be used for follow-up of patients to determine their potential as a prognostic test for malignant transformation.

Expression profile of malignant and non-malignant diseases of the thyroid gland reveals altered expression of a common set of genes in goiter and papillary carcinomas.

Using cDNA microarrays with 3800 cDNA fragments, we determined the expression profile of normal thyroid tissue, goiter, adenoma and papillary carcinoma (10 samples from each class). After background correction and statistical analysis, we identified a set of 160 genes as being differentially expressed in all pair-wise comparisons. Here we demonstrate that, at least on the basis of these differentially expressed genes, a positive correlation between goiter and papillary carcinomas could be observed. We identified a common set of genes whose expression is diminished in both goiter and papillary carcinomas as compared to normal thyroid tissue. Moreover, no genes with inverse correlation in samples from goiter and papillary carcinomas could be detected. Using Real-Time PCR and/or tissue microarrays, we confirmed the altered expression of some of the identified genes. Of notice, we demonstrate that the reduced mRNA levels of p27(kip1) observed in papillary carcinomas as compared to either goiter or normal thyroid tissues (P<0.001) is accompanied by an altered protein distribution within the cell. In papillary carcinomas, P27(KIP1) is preferentially cytoplasmic as opposed to goiter or normal thyroid tissue, where P27(KIP1) is preferentially located in the nucleus. The exploitation of the data presented here could contribute to the understanding of the molecular events related to thyroid diseases and gives support to the notion that common molecular events might be related to the frequent observation of areas of papillary carcinomas in the gland of patients with goiter.

Differential expression of IGFBP-5 and two human ESTs in thyroid glands with goiter, adenoma and papillary or follicular carcinomas.

Here, we describe the identification of three human genes with altered expression in thyroid diseases. One of them corresponds to insulin-like growth factor binding protein 5 (IGFBP5), which has already been described as over expressed in other cancers and, for the first time, is identified as overexpressed in thyroid tumors. The other genes, named 44 and 199, are ESTs with yet unknown function and were mapped on human chromosomes seven and four, respectively. We determined by RT-PCR the expression level of these genes in ten samples of disease-free thyroid, ten of goiter, nine of papillary carcinoma, ten of adenoma and seven of follicular carcinoma and the significance of observed differences was statistically determined. IGFBP-5 and gene 44 were significantly overexpressed in papillary carcinoma when compared to normal and goiter. Genes 44 and 199 were differentially expressed in follicular carcinoma and adenoma when compared to normal thyroid tissue.

Differentially expressed genes in gastric tumors identified by cDNA array.

Using cDNA fragments from the FAPESP/lICR Cancer Genome Project, we constructed a cDNA array having 4512 elements and determined gene expression in six normal and six tumor gastric tissues. Using t-statistics, we identified 80 cDNAs whose expression in normal and tumor samples differed more than 3.5 sample standard deviations. Using Self-Organizing Map, the expression profile of these cDNAs allowed perfect separation of malignant and non-malignant samples. Using the supervised learning procedure Support Vector Machine, we identified trios of cDNAs that could be used to classify samples as normal or tumor, based on single-array analysis. Finally, we identified genes with altered linear correlation when their expression in normal and tumor samples were compared. Further investigation concerning the function of these genes could contribute to the understanding of gastric carcinogenesis and may prove useful in molecular diagnostics.

Trypanosoma cruzi gene expression in response to gamma radiation.

Trypanosoma cruzi is an organism highly resistant to ionizing radiation. Following a dose of 500 Gy of gamma radiation, the fragmented genomic DNA is gradually reconstructed and the pattern of chromosomal bands is restored in less than 48 hours. Cell growth arrests after irradiation but, while DNA is completely fragmented, RNA maintains its integrity. In this work we compared the transcriptional profiles of irradiated and non-irradiated epimastigotes at different time points after irradiation using microarray. In total, 273 genes were differentially expressed; from these, 160 were up-regulated and 113 down-regulated. We found that genes with predicted functions are the most prevalent in the down-regulated gene category. Translation and protein metabolic processes, as well as generation of precursor of metabolites and energy pathways were affected. In contrast, the up-regulated category was mainly composed of obsolete sequences (which included some genes of the kinetoplast DNA), genes coding for hypothetical proteins, and Retrotransposon Hot Spot genes. Finally, the tyrosyl-DNA phosphodiesterase 1, a gene involved in double-strand DNA break repair process, was up-regulated. Our study demonstrated the peculiar response to ionizing radiation, raising questions about how this organism changes its gene expression to manage such a harmful stress.

Early changes in gene expression induced by tobacco smoke: Evidence for the importance of estrogen within lung tissue.

Lung cancer is the leading cause of cancer deaths in the United States, surpassing breast cancer as the primary cause of cancer-related mortality in women. The goal of the present study was to identify early molecular changes in the lung induced by exposure to tobacco smoke and thus identify potential targets for chemoprevention. Female A/J mice were exposed to either tobacco smoke or HEPA-filtered air via a whole-body exposure chamber (6 h/d, 5 d/wk for 3, 8, and 20 weeks). Gene expression profiles of lung tissue from control and smoke-exposed animals were established using a 15K cDNA microarray. Cytochrome P450 1b1, a phase I enzyme involved in both the metabolism of xenobiotics and the 4-hydroxylation of 17beta-estradiol (E(2)), was modulated to the greatest extent following smoke exposure. A panel of 10 genes were found to be differentially expressed in control and smoke-exposed lung tissues at 3, 8, and 20 weeks (P < 0.001). The interaction network of these differentially expressed genes revealed new pathways modulated by short-term smoke exposure, including estrogen metabolism. In addition, E(2) was detected within murine lung tissue by gas chromatography-coupled mass spectrometry and immunohistochemistry. Identification of the early molecular events that contribute to lung tumor formation is anticipated to lead to the development of promising targeted chemopreventive therapies. In conclusion, the presence of E(2) within lung tissue when combined with the modulation of cytochrome P450 1b1 and other estrogen metabolism genes by tobacco smoke provides novel insight into a possible role for estrogens in lung cancer.

Characterization of global transcription profile of normal and HPV-immortalized keratinocytes and their response to TNF treatment.

BACKGROUND: Persistent infection by high risk HPV types (e.g. HPV-16, -18, -31, and -45) is the main risk factor for development of cervical intraepithelial neoplasia and cervical cancer. Tumor necrosis factor (TNF) is a key mediator of epithelial cell inflammatory response and exerts a potent cytostatic effect on normal or HPV16, but not on HPV18 immortalized keratinocytes. Moreover, several cervical carcinoma-derived cell lines are resistant to TNF anti-proliferative effect suggesting that the acquisition of TNF-resistance may constitute an important step in HPV-mediated carcinogenesis. In the present study, we compared the gene expression profiles of normal and HPV16 or 18 immortalized human keratinocytes before and after treatment with TNF for 3 or 60 hours. METHODS: In this study, we determined the transcriptional changes 3 and 60 hours after TNF treatment of normal, HPV16 and HPV18 immortalized keratinocytes by microarray analysis. The expression pattern of two genes observed by microarray was confirmed by Northern Blot. NF-kappaB activation was also determined by electrophoretic mobility shift assay (EMSA) using specific oligonucleotides and nuclear protein extracts. RESULTS: We observed the differential expression of a common set of genes in two TNF-sensitive cell lines that differs from those modulated in TNF-resistant ones. This information was used to define genes whose differential expression could be associated with the differential response to TNF, such as: KLK7 (kallikrein 7), SOD2 (superoxide dismutase 2), 100P (S100 calcium binding protein P), PI3 (protease inhibitor 3, skin-derived), CSTA (cystatin A), RARRES1 (retinoic acid receptor responder 1), and LXN (latexin). The differential expression of the KLK7 and SOD2 transcripts was confirmed by Northern blot. Moreover, we observed that SOD2 expression correlates with the differential NF-kappaB activation exhibited by TNF-sensitive and TNF-resistant cells. CONCLUSION: This is the first in depth analysis of the differential effect of TNF on normal and HPV16 or HPV18 immortalized keratinocytes. Our findings may be useful for the identification of genes involved in TNF resistance acquisition and candidate genes which deregulated expression may be associated with cervical disease establishment and/or progression.

Class distinction between follicular adenomas and follicular carcinomas of the thyroid gland on the basis of their signature expression.

BACKGROUND: Nodules of the thyroid gland are observed frequently in patients who undergo ultrasound studies. The majority of these nodules are benign, corresponding to goiters or adenomas, and only a small fraction corresponds to carcinomas. Among thyroid tumors, the diagnosis of follicular adenocarcinomas by preoperative fine-needle aspiration biopsy is a major challenge, because it requires inspection of the entire capsule to differentiate it from adenoma. Consequently, large numbers of patients undergo unnecessary thyroidectomy. METHODS: Using data from gene expression analysis, the authors applied Fisher linear discriminant analysis and searched for expression signatures of individual samples of adenomas and follicular carcinomas that could be used as molecular classifiers for the precise classification of malignant and nonmalignant lesions. RESULTS: Fourteen trios of genes were described that fulfilled the criteria for the correct classification of 100% of samples. The robustness of these trios was verified by using leave-1-out cross-validation and bootstrap analyses. The results demonstrated that, by combining trios, better classifiers could be generated that correctly classified >92% of samples. CONCLUSIONS: The strategy of classifiers based on individual signatures was a useful strategy for distinguishing between samples with very similar expression profiles.

Gene expression profiles in breast tumors regarding the presence or absence of estrogen and progesterone receptors.

Estrogen acts via its receptor (ER) to stimulate cell growth and differentiation in the mammary gland. ER and progesterone receptor (PR), which is regulated by estrogen via ER, have been used as prognostic markers in clinical management of breast cancer patients. Patients with ER- breast tumors have a poorer prognosis than patients with ER+ tumors. The aim of the present study was the identification of tumor-associated genes differentially expressed in breast tumors regarding the presence or absence of ER and PR hybridized with cDNA microarrays containing 4,500 tumor-derived expressed sequence tags generated using the ORESTES technique. Samples of human primary breast carcinomas from 38 patients were analyzed. The experiments were performed in triplicates and data from each element were acquired by phosphoimage scanning. Data acquisition was performed using the ArrayVision software. After normalization statistical analysis was applied. In a preliminary analysis, 98 differentially expressed transcripts were identified, 46 were found to be more expressed in ER+/PR+ and 52 were found to be more expressed in ER-/PR- breast tumors. The biochemical functions of the genes in the reported expression profile are diverse and include metabolic enzymes, protein kinases, helicases, transcription factors, cell cycle regulators and apoptotic factors. ER-/PR- breast tumors displayed increased levels of transcripts of genes associated with neurodegeneration and genes associated with proliferation were found in ER+/PR+ tumors.

Comparative analysis of amplified and nonamplified RNA for hybridization in cDNA microarray.

Limiting amounts of RNA is a major issue in cDNA microarray, especially when one is dealing with fresh tissue samples. Here we describe a protocol based on template switch and T7 amplification that led to efficient and linear amplification of 1300x. Using a glass-array containing 368 genes printed in three or six replicas covering a wide range of expression levels and ratios, we determined quality and reproducibility of the data obtained from one nonamplified and two independently amplified RNAs (aRNA) derived from normal and tumor samples using replicas with dye exchange (dye-swap measurements). Overall, signal-to-noise ratio improved when we used aRNA (1.45-fold for channel 1 and 2.02-fold for channel 2), increasing by 6% the number of spots with meaningful data. Measurements arising from independent aRNA samples showed strong correlation among themselves (r(2)=0.962) and with those from the nonamplified sample (r(2)=0.975), indicating the reproducibility and fidelity of the amplification procedure. Measurement differences, i.e, spots with poor correlation between amplified and nonamplified measurements, did not show association with gene sequence, expression intensity, or expression ratio and can, therefore, be compensated with replication. In conclusion, aRNA can be used routinely in cDNA microarray analysis, leading to improved quality of data with high fidelity and reproducibility.