Gene expression profiles of primary HPV16- and HPV18-infected early stage cervical cancers and normal cervical epithelium: identification of novel candidate molecular markers for cervical cancer diagnosis and therapy

Abstract

With the goal of identifying genes with a differential pattern of expression between invasive cervical carcinomas (CVX) and normal cervical keratinocytes (NCK), we used oligonucleotide microarrays to interrogate the expression of 14,500 known genes in 11 primary HPV16 and HPV18-infected stage IB-IIA cervical cancers and four primary normal cervical keratinocyte cultures. Hierarchical cluster analysis of gene expression data identified 240 and 265 genes that exhibited greater than twofold up-regulation and down-regulation, respectively, in primary CVX when compared to NCK. Cyclin-dependent kinase inhibitor 2A (CDKN2A/p16), mesoderm-specific transcript, forkhead box M1, v-myb myeloblastosis viral oncogene homolog (avian)-like2 (v-Myb), minichromosome maintenance proteins 2, 4, and 5, cyclin B1, prostaglandin E synthase (PTGES), topoisomerase II alpha (TOP2A), ubiquitin-conjugating enzyme E2C, CD97 antigen, E2F transcription factor 1, and dUTP pyrophosphatase were among the most highly overexpressed genes in CVX when compared to NCK. Down-regulated genes in CVX included transforming growth factor beta 1, transforming growth factor alpha, CFLAR, serine proteinase inhibitors (SERPING1 and SERPINF1), cadherin 13, protease inhibitor 3, keratin 16, and tissue factor pathway inhibitor-2 (TFPI-2). Differential expression of some of these genes including CDKN2A/p16, v-Myb, PTGES, and TOP2A was validated by quantitative real-time PCR. Flow cytometry on primary CVX and NCK and immunohistochemical staining of formalin fixed paraffin-embedded tumor specimens from which primary CVX cultures were derived as well as from a separate set of invasive cervical cancers confirmed differential expression of the CDKN2A/p16 and PTGES markers on CVX versus NCK. These results identify several genes that are coordinately disregulated in cervical cancer, likely representing common signaling pathways triggered by HPV transformation. Moreover, these data obtained with highly purified primary tumor cultures highlight novel molecular features of human cervical cancer and provide a foundation for the development of new type-specific diagnostic and therapeutic strategies for this disease.

mRNA biomarker detection in liquid-based cytology: a new approach in the prevention of cervical cancer

“…18,19 In our study, the analysis of these biomarkers in biopsy material by immunohistochemistry showed that all were positive in a high proportion of the HSIL lesions, which is consistent with current evidence. 8,16,[20][21][22][23] Nevertheless, immunohistochemistry is not adequate for screening purposes. Finally, immunohistochemistry only analyzes a small area of the lesion, which may not be representative of the whole process that could explain the absence of correlation between the mRNA expression in the liquid-based cytology and the immunohistochemistry staining in the simultaneous biopsy for most of the biomarkers.…”

“…The list of candidates who have shown potential utility in cervical cancer screening includes p16 INK4a (CDKN2A), survivin (BIRC5), metalloproteinase 9 (MMP9), topoisomerase 2 alpha (TOP2A), minichromosome maintenance 5 (MCM5), or MKi67 proteins (MKI67). [7][8][9] Most studies focused on these biomarkers have used immunohistochemistry to detect protein expression. However, several shortcomings of immunohistochemistry, in particular the interobserver variation inherent to all morphological techniques and the difficulty to obtain reproducible quantification, hamper proper evaluation of the clinical usefulness of these biomarkers.…”

Gene expression reveals two distinct groups of anal carcinomas with clinical implications

“…However, the effect of high-risk HPV on global gene expression has been studied and characterised in several model systems on different microarray platforms (Chang and Laimins, 2000;Nees et al, 2001;Alazawi et al, 2002;Vernell et al, 2003;Garner-Hamrick et al, 2004;Loercher et al, 2004;Santin et al, 2005). Several biomarkers predicting both HPV status and response to treatment for several cancers have been proposed.…”

“…The resulting hierarchical clustering (Figure 3Ad) was highly similar to both the hierarchical clustering of our unsupervised analysis (Figure 3Aa) and to the hierarchical clustering of genes selected by performing two independent statistical analyses between the two tumour groups (Figure 3Ab and Ac). Similarly, to investigate the possible transcriptional influence of high-risk HPV, we performed hierarchical cluster analysis on genes reported differentially Figure 3 (A) Hierarchical clustering of the unsupervised analysis on (Aa) the selection of 8449 probes, (Ab) the 337 individual genes defined by the Ttest, (Ac) the 363 individual genes defined by the SAM analysis together with collections of genes described by others relevant to our study and included within the group of 8449 probes, (Ad) HPV16-induced gene expression (Alazawi et al, 2002), (Ae) HPV16 E6/E7-induced gene expression (Nees et al, 2001), (Af) HPV31-induced gene expression (Chang and Laimins, 2000), (Ag) Gene expression in SSC (Loercher et al, 2004), (Ah) E2F-regulated genes (Ishida et al, 2001), (Ai) HPV16-and HPV18-induced gene expression (Santin et al, 2005) and, finally, (Aj) HPV18 E6/E7-induced gene expression (GarnerHamrick et al, 2004). (B) The total number of genes within each report cited.…”

“…(C) Hierarchical cluster analysis based on (Ca) CDKN2A (p16) transcriptionally regulated genes and (Cb) E2F-regulated genes, as reported by Vernell et al, 2003. expressed in several systems related to high-risk HPV infection. This included gene expression profiling of a mouse SCC model (Loercher et al, 2004), effect on overall gene expression associated with integration of HPV16 in cervix SCC (Alazawi et al, 2002), gene expression profiling of primary HPV16-and HPV18-infected early stage cervical cancer (Santin et al, 2005), effect of HPV18 E6 and E7 on global gene expression in an organotypic keratinocytes culture system (Garner-Hamrick et al, 2004), effect of HPV16 E6 and/or E7 on global gene expression in differentiating cervical keratinocytes (Nees et al, 2001) and, finally, effect of HPV31 on global gene expression in normal human keratinocytes (Chang and Laimins, 2000). Again, the resulting hierarchical cluster (Figure 3Ae-Aj) was practically identical compared to our unsupervised analysis ( Figure 3Aa).…”