Table 1.
Patients followed up for at least 42 months for survival evaluation.
Figure 1.
Distribution of deregulated genes according to the fold change (FC) and Δ-score values.
All 997 genes (circles) that were deregulated in cervical cancer (CC) tumors compared to the control samples by the SAM method are graphed in a Volcano plot. The x-axis represents the FC in gene expression (cancer sample/control sample) expressed in Log2 and the y-axis display the absolute Δ-score, a modified t-test calculated with the SAM method, the higher the Δ-score values, the higher the statistical significance. The Log2 (FC) values are positive for upregulated genes and negative for downregulated genes. Circles colored in red and orange represent the genes involved in M-phase of the cell cycle and those colored in blue and orange are the genes that were validated by qRT-PCR.
Figure 2.
Segregation of tumor and control samples according to the expression of deregulated genes.
Unsupervised hierarchical cluster analysis of 43 CCs and 12 healthy cervical epitheliums using the expression values obtained with the HG-Focus microarray of all 997 deregulated genes (panel A) or the 23 top ranked genes selected for validation (panel B). Each row represents a gene and each column represents a sample. 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, green for downregulation, and black for unchanged expression. Panel C shows the principal components analysis (PCA) using the values in panel B; blue circles represent the CCs (n = 43) and yellow circles represent the controls (n = 12). Both sets of genes clearly separated the samples into the 2 main groups using both types of analysis.
Table 2.
Genes explored by qRT-PCR.
Figure 3.
Correlation of expression intensity of 23 genes examined by HG-Focus and HG-ST1.0 microarrays.
The Log2 values of the standardized intensity signals (RMA values) of 23 genes examined by the 2 microarrays in 19 CC and 5 normal cervical epithelium were plotted. The linear trend (black line) is included, which was calculated with Person’s correlation test. r = correlation coefficient, p = p-value.
Figure 4.
Validation of gene expression of 9 genetic markers by qRT-PCR.
The intensity of gene expression, expressed in Log2 values, is shown in box plots. Expression of the 6 genes validated in this study (CCNB2, PRC1, SYCP2, CDKN3, CDC20, and NUSAP1) and the 3 well-known genes (CDKN2A, MKI67, and PCNA) associated with CC are compared among the 4 groups, including healthy cervical epitheliums (Normal, n = 25), low-grade CIN (CIN1, n = 29), high-grade CIN (CIN2/3, n = 21), and invasive CC (cancer, n = 44). The upper and lower boundaries of the boxes represent the 75th and 25th percentiles, respectively. The black line within the box represents the median value, and the whiskers represent the minimum and maximum values that lie within 1.5× the interquartile range from the end of box. Values outside this range are represented by black circles. The fold change (FC) was calculated by dividing the median of each pathological group by the median of the control group.
Table 3.
ROC analysis and calculus of sensitivity, specificity and predictive values.
Table 4.
ROC analysis of 4 gene markers selected for detection of CIN2/3 and CC.
Figure 5.
Histological analysis of marker genes.
Protein expression was determined by immunohistochemistry using sections from formalin-fixed, paraffin-embedded tissue. Proteins explored were CDKN3A, SYCP2, PRC1, CDC20, CCNB2, PCNA, CDKN2A, MKI67, and CDC2. Representative experiments in adeno cell carcinomas (panel A) and squamous cell carcinomas (panel B) are shown. The specific signals are shown as brown staining (counterstained with hematoxylin; original magnification, ×400; bars, 10 µm).
Figure 6.
Percentage of tumor cells with positive signals for the markers tested by immunohistochemistry.
The average percentages of tumor cells with positive signals in the nucleus (panel A) or cytoplasm (panel B) for CDC20, CCNB2, CDC2, CDKN2A, CDKN3, MKI67, PCNA, PRC1, and SYCP2 are plotted. Tumors positive for HPV16 (blue bars) are compared with tumors positive for other HPVs (red bars). As NUSAP1 was only explored in HPV16-positive CCs, the average percentages of tumor cells with positive signals in nuclei (32.5%) and cytoplasm (68.4%) were not included in this figure. The numbers for the entire set of tumors is also included (green bars). The standard error is shown.
Figure 7.
Survival analysis of women with cervical cancer according to FIGO staging and CDKN3 expression.
The Kaplan-Meier curves for FIGO staging and CDKN3 are shown. Patients were followed-up for at least 42 months. For gene expression, cancer patients with higher (red line) and lower (blue line) fold change values were compared (see material and methods). The p value was calculated by comparing the curves with the log-rank test. Censored patients are labeled with black dots, but only four of them were censored before the minimal period of follow-up (42 months).
Figure 8.
Canonical pathways where deregulated genes are involved.
Top 25 canonical pathways identified in the set of 997 deregulated genes between the tumors and controls (A) and in the subset of the 100 best-ranked genes (50 upregulated and 50 downregulated; B). The canonical pathways were identified using the Ingenuity Pathway Analysis (IPA) system. The −log (p-value) (blue bars) and the ratio (yellow dots) were calculated by comparing the number of genes in the pathways present in the datasets versus the human database. The p-value was calculated using the chi square test or Fisher’s exact test as appropriate, and the -log (p-value) values >1.3 (red line) correspond to p<0.05.
Table 5.
DAVID functional annotation cluster analysis at the highest stringency of 997 genes deregulated in cervical cancer*.
Table 6.
DAVID functional annotation cluster analysis at the highest stringency of the 100 genes most deregulated in cervical cancer compared with normal cervical epithelium*.