Patients followed up on average for 63 months for survival evaluation.

a<p>ACC, Adenocarcinoma. SCC, Squamous Cell Carcinoma. ASCC, Adenosquamous Cell Carcinoma.</p><p>bHT, Radical Hysterectomy. Tele, teletherapy. Brachy, brachytherapy. Chemo, chemotherapy with Cisplatin. i. Means incomplete treatment.</p>c<p>Status alive at the last follow up record and death was caused by primary tumor of cervical cancer, except the case labeled with an asterisk. The cause of death was unknown.</p>d<p>CN indicate the samples analyzed for CN (500 K array), CN/EX indicate the samples analyzed for CN (500 K array) and gene expression (HG 1.0 ST array), and EX indicate the samples analyzed for gene expression (HG 1.0 ST).</p

DAVID functional annotation cluster analysis in the 2006 genes differentially expressed in cervical cancer.

<p>*The cluster number was obtained when the analysis was run with the whole gene set, including up- and down-regulated genes.</p><p>FC = Fold change is the ratio of the proportion of genes in the tested list versus the Human Gene Reference database.</p><p>NC =  No clustered in up (+) and down (−) regulated genes analyzed separately.</p><p>The clusters in italics were enriched when the functional annotation cluster analysis was run at the highest stringency, and the number inside the parenthesis indicated the order the cluster occupied in the list.</p

Segregation of tumors and control samples according to gene expression profile.

<p>Unsupervised hierarchical cluster analysis of 55 CCs and 17 healthy cervical epithelium samples using the expression values of the genes deregulated from glycolysis (panel A) and the anaphase-promoting complex/cyclosome (APC/C)-dependent proteasomal protein catabolic process (panel B) obtained with the HG 1.0 ST microarray. Each row represents a gene and each column represents a sample. Samples name beginning with an “R” are CCs and with a “C” are controls; CCs ending in 1, 2 or 3 belong to low-, medium- or high-CN groups, respectively, whereas those ending with no number were not explored for CN. The length and the subdivision of the branches represent the relationships among the samples based on the intensity of gene expression. The cluster is color-coded using red for upregulation, blue for downregulation, and white for unchanged expression. In panel B, the sub-branches enclosed in squares were considered together in a group with a strong upregulation profile and the remaining upregulated samples were placed in another group with a weak upregulation profile.</p

Comparison between CN and gene expression by chromosome arm.

<p>The left side shows the explored genome (Mb) in each chromosome arm that was explored with the 500 K microarray in 31 tumors. The right side shows the number of genes on each arm explored with the Human Gene 1.0 ST microarray in 27 of the tumors in which CN was examined. Each bar represents the percentage of CN-AG (left) or deregulated genes (right) of the chromosomal arm indicated in the middle. Red bars indicate gains or overexpression and blue bars represent losses or subexpression. The dotted line represents the average percentage of the global CN-AG (8.1%; left) or the percentage of deregulated genes (9.5%; right) in the tumor genome. The arms marked with asterisks had an average proportion of CN-AG or percentage of deregulated genes superior and statistically significant compared to the numbers found in the complete tumor genome (p<0.05, chi-square).</p

Survival analysis of women with CC according to International Federation of Gynecology and Obstetrics (FIGO) staging, CN-AG, and gene expression profiles.

<p>The Kaplan-Meier curves for FIGO staging, the whole and 3q %CN-AG, and gene expression profiles of genes involved in glycolysis and APC/C-dependent proteasomal protein catabolic process (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097842#pone-0097842-g009" target="_blank">Figure 9</a>) are shown. Patients were followed up an average of 63 months. The p value was calculated by comparing the curves with the log-rank test. Censored patients are labeled with transverse lines.</p

Validation of the GeneChip Human Mapping 500(500 K) microarray with the high-density CytoScan HD microarray.

<p>Intensity signals of single nucleotide polymorphisms (SNPs) or non-polymorphic probes are expressed as log₂ ratios from chromosomes 3, 4, and 5 of the tumor R496 explored using the 500 K microarray (upper panel) and HD 2.7 microarray (lower panel). In both panels, the y-axis depicts a log₂ ratio scale from –1.5 to 1.5, and the x-axis shows the ideogram of the explored chromosomes with genome positions. The horizontal line crossing the point y = 0 corresponds to 2 copies. The average density of explored positions is more than 5 times higher in the HD 2.7 microarray than that in the 500 K microarray (see materials and methods).</p

Analysis workflow of 59 cervical cancer cases.

<p>Figure shows the analysis workflow of the 59 CC cases explored in this study. All CC samples were HPV16 positive and were investigated with microarrays–31 for mapping CN-AG and 55 for global gene expression, with 27 CCs in common. These 27 CCs were used for the analyses of global gene expression and the correlation between the CN-AG and gene expression. For the hierarchical clustering analysis, the expression profiles of the 55 CC samples were included. Five-year survival was investigated in 55 patients, 51 explored for gene expression and 28 for CN alterations, with 24 CCs in common. See material and methods section for details of the procedures.</p

Validation of genes amplified and deregulated located in 3q by qPCR and qRT-PCR.

<p>The top panel shows the average copy number of 7 genes (<i>CLDN1</i>, <i>ECT2</i>, <i>NAALADL2</i>, <i>NLGN1</i>, <i>PLOD2</i>, <i>PLSCR1</i>, and <i>PLSCR4</i>) located in 3q explored with qPCR in controls (lymphocytes) and tumors with 2 or 3–4 copies identified with the 500 K microarray. Whiskers of each bar represent the standard error of the mean. The dotted red line shows the value for 2.5 copies calculated with qPCR (see material and methods). The bottom panel shows the correlation of gene expression of 8 genes (<i>MCM2</i>, <i>PLOD2</i>, <i>PLSCR1</i>, <i>SMC4</i>, <i>ECT2</i>, <i>NLGN1</i>, <i>RFC4</i>, and <i>CLDN1</i>) located in 3q explored in 27 tumors and 6 controls with both the HG 1.0 ST microarray and qRT-PCR techniques. Log₂ values of the normalized intensity signals obtained with the microarray (robust multichip average values) and qRT-PCR were plotted. Trend line (black line), correlation coefficient (r), and p value were calculated with Pearson’s correlation test.</p

Influence of copy number alterations in gene deregulation in cervical carcinomas.

a<p>21,034 genes were explored in all but four tumors (R062, R111, R116, R248) for changes in expression with HG 1.0 ST microarray. On average 1,673 of them were CN-altered (CN1/CN3) and 19,362 did not have copy number alterations (CN2). CN1 means genes with one copy deleted, CN2 means genes without CN alterations (2 copies), and CN3 means amplified genes with three or four copies.</p>b<p>Copy number altered genes according to data obtained with 500 K microarray.</p><p>n = number of genes identified in the analysis of copy number with 500 K microarray that were also explored for gene expression with the HG 1.0 ST microarray.</p><p>EX+ = Genes that were up- or down- regulated in tumors compared with the control sample.</p><p>Samples R075 and R189 are ACC and R298 is ASCC, the rest are SCC.</p><p>Samples labeled with an asterisk were excluded for the survival analysis.</p

Identification of CN-altered genes in biological processes enriched in high-CN tumors.

<p>Shown are the numbers of 2-copy or CN-altered genes in ≤3 tumors (green bars) and CN-altered genes in ≥4 tumors (blue bars) among the biological processes enriched in the subset of genes deregulated exclusively in high-CN tumors.</p