We examined the status of promoter hypermethylation of 16 genes (CDH1, DAPK, RARB, HIC1, FHIT, RASSF1A, APC, CDKN2A, MGMT, BRCA1, TP73, TIMP3, GSTP1, MLH1, p14^ARF, and RB1) in eight specimens of normal cervical squamous epithelia and 90 CC specimens. Promoter hypermethylation was not found in the DNA isolated from normal cervical smears for any of the tested genes. However, hypermethylation was detected in one or more genes in 79 of 90 (87.8%) CC specimens. The frequency of promoter hypermethylation for individual genes was: CDH1, 51.1%; DAPK, 43.3%; RARB, 33.3%; HIC1, 22.2%; FHIT, 11.1%; RASSF1A, 10%; APC, 10%; CDKN2A, 8.9%; MGMT, 6.7%; BRCA1, 5.6%; TP73, 2.2%; TIMP3, 1.1%; GSTP1, 1.1%; and MLH1, 1.1% (Table 1). The remaining two genes (p14 ^ARF and RB1) did not show promoter methylation. Seventy-one of 82 (86.6%) primary tumors and 8 of 8 (100%) cell lines exhibited methylation. Although the patterns are similar, primary tumors had a higher frequency of methylation of CDH1 and DAPK genes as compared to cell lines, while RARB, HIC1, RASSF1A, MGMT, and TP73 genes had higher prevalence in cell lines as compared to primary tumors (Table 1). A high frequency of promoter methylation was detected in primary tumors for CDH1 (54.9%; 45 of 82 tumors) and DAPK (45.1%; 37 of 82 tumors). RARB (29.3%) and HIC1 (18.3%) genes were less frequently methylated in primary tumors. Other genes, FHIT (11%), APC (11%), CDKN2A (8.5%), RASSF1A (7.3%), BRCA1 (6.1%), MGMT (4.9%), and TIMP3 (1.2%), were rarely methylated in primary CC (Table 1, Fig. 1).

Eighty-six of 90 (95.6%) tumors were positive for HPV DNA. The remaining four tumors were HPV negative. HPV 16 alone or in combination with other HPV types was found in 55 (61.1%). HPV 18 was found in eight (8.9%) tumors, while other HPV types (20 different types) in 23 (25.6%) tumors.

Of the 82 primary tumors studied for promoter methylation, 48 tumors were also examined for microsatellite instability (MSI) using 49 di- or tetra-nucleotide markers 14151617. We found 9 of 48 (18.8%) tumors had MSI-H, and 9 of 48 (18.8%) tumors exhibited MSI-L phenotype. The remaining tumors did not show MSI (Table 2).

To evaluate the relationship between promoter hypermethylation and clinico-pathologic features, as well as other genetic changes, methylation status was correlated with histology, age, stage and size of the tumor, clinical outcome, HPV type, and microsatellite instability. Table 2 shows the results of univariate analyses examining the associations between methylation and these characteristics. Although both squamous cell carcinoma (SCC; 85.7%) and adenocarcinoma (AC; 100%) exhibited an overall high rate of promoter methylation, we found statistically significant differences in various genes in these two histologic subsets (Table 2). Promoter methylation of the CDH1, RARB, FHIT, and MGMT genes was noted only in SCC, whereas the AC exhibited significantly higher frequency of promoter methylation of HIC1 (60% in AC vs. 15.6% in SCC), APC (80% in AC vs. 6.5% in SCC), and BRCA1 (40% in AC vs. 3.9% in SCC) (Table 2).

A significant correlation was observed between the overall frequency of methylation and the tumor stage (P = 0.001). Higher stage (stages II-IV) tumors exhibited an increased overall frequency of promoter methylation (Table 2). However, the differences were not statistically significant when individual genes examined. On the other hand, RASSF1A methylation was associated with low-grade tumors (P = 0.003). Tumor size has no effect on the overall frequency of methylation. Age also has no influence on overall frequency of promoter methylation. However, RARB gene was more frequently methylated in younger patients (34.7% in below 50 years and 21.2% in above 50 years age) (P = 0.02). Promoter methylation of APC (2.0% in patients below 50 years vs. 24.2% in patients above 50 years age) (P= 0.001) and BRCA1 (4.1% in below 50 years and 9.1% in patients above 50 years) (P = 0.05) genes were significantly higher in older patients.

In order to assess the prognostic role, we also correlated treatment response and clinical outcome with promoter methylation. The overall frequency of methylation was higher in patients who died of cancer (DOC) (95.3%) as compared to those who were alive with recurring disease or complete remission (76.9%) (Table 2) (P = 0.015). Although statistically not significant, all the patients whose tumors showed promoter methylation of MGMT (4 tumors) and BRCA1 (5 tumors) genes DOC within 1–21 months of follow-up after the initial diagnosis. Promoter methylation of the remaining genes did not show such differences in relation to the outcome. To assess the role of radiation and/or chemotherapy treatment on outcome, we correlated methylation with response in patients with at least 5 months follow up after treatment. No significant differences in the overall promoter methylation were found in patients with complete remission (CR) compared to patients who DOC or had only partial response (PR). In this group, all the patients that showed methylation of MGMT and BRCA1 genes failed to respond to the treatment suggesting promoter methylation of these genes may be a bad prognostic indicator (Table 2). Promoter methylation of RARB gene was also predicted a worse prognosis as 80% of the patients with methylation were either DOC or only partial responders compared to only 20% patients with CR exhibited methylation of this gene (Table 2).

Table 3 summarizes the results from multivariate Cox Survival models. Overall, after adjusting for age, treatment, tumor size, stage and HPV type, neither total number of methylated genes nor presence of methylation of each specific gene were statistically significantly related to survival. However, methylation of 3 genes did suggest a doubling (RASSF1A and MGMT) or tripling (BRCA1) of risk compared to those who did not have these genes methylated. These differences were not statistically significant due to small number of tumors analyzed and need to be confirmed using a larger sample size.

Type of HPV infection had no association with the status of promoter methylation (Table 2). Although the MSI phenotype also had no significant correlation with overall promoter methylation, the HIC1 (P = 0.02) and APC (P = 0.08) genes were frequently methylated in patients exhibiting MSI-H phenotype (Table 2).

To examine the role of CDH1 and RARB genes in CC progression, we carried out immunohistochemical analysis in 39 CIN specimens (Nine low-grade and 30 high-grade). None of the low-grade CINs showed down-regulated expression, while only 2 of 30 (6.7%) showed absent or reduced expression suggesting that CDH1 inactivation is a later event in CC development. The expression pattern of RARB in normal epithelium is complex since the staining intensities are highly variable between nuclear and cytoplasmic compartments. There was no normal epithelium to evaluate the expression in most CIN cases studied. However, all the cases that we evaluated for RARB immunoreactivity showed some positive mesenchymal cells. We evaluated the intensity of nuclear staining in 34 cases of paraffin-embedded tissues. One (11.1%) of the nine low-grade CINs showed decreased nuclear expression, whereas, 15 (60%) of 25 evaluable high-grade CIN lesions showed complete lack of nuclear expression (Fig. 2). If confirmed, the down-regulated RARB expression in high-grade CINs suggests that this occurs early in progression and plays a role in the development of CC.

To examine the biological role of promoter methylation in CC, we assessed the levels of mRNA by semi-quantitative RT-PCR in eight cell lines and compared with the respective control values obtained from the averages calculated from 2 normal cervix after normalization against ACTB. This analysis was performed on 7 genes (CDH1, DAPK, RARB, HIC1, MGMT, RASSF1A, and MLH1) that exhibited methylation in one or more cell lines and primary tumors (Fig. 3). RARB gene showed either complete absence of or down regulated expression in all cell lines (6 methylated and 2 unmethylated). Of the six cell lines with RARB gene methylated, three (HT-3, MS751, and ME-180) exhibited reactivation upon treatment with 5-Aza-2' deoxycytidine or n-butyrate alone or the combination of the two. Three cell lines (C4-I, CaSki, and SW756) that had promoter methylation of RARB did not reactivate expression after treatment with these drugs (Fig. 4). One of the two unmethylated cell lines also reactivated the expression of RARB.

The CDH gene exhibited a complete absence of expression in one cell line (SW756) that showed promoter hypermethylation and two other cell lines (C-33A and SiHa) without evidence of methylation (Fig. 3). The SW756 cell line with methylation and one of the two unmethylated cell lines showed reactivated expression upon treatment with 5-Aza-2' deoxycytidine or in combination with n-butyrate, but not with n-butyrate alone. The remaining five cell lines neither showed evidence of decreased mRNA nor any effect of demethylating or HDAC-inhibiting agents on expression levels.

The DAPK gene showed a complete absence of expression in five and down regulated expression in one of the 8 CC cell lines studied (Fig. 3). Of the six cell lines with down regulated expression, 2 had promoter methylation and 4 had unmethylated promoter of the DAPK gene. Both methylated cell lines (HT-3 and SiHa) and three (C-33A, MS751, and ME-180) of the four unmethylated cell lines showed reactivation upon exposure to 5-Aza-2' deoxycytidine or in combination with n-butyrate. One of the unmethylated cell lines (SW756) that showed a complete absence of expression did not reactivate after treatment.

The HIC1 gene was methylated in 5 of 8 cell lines. Three of the methylated cell lines (C-4I, SiHa, and ME-180) and one of the three unmethylated cell lines (MS751) showed down regulated expression. Treatment of 5-Aza-2' deoxycytidine or n-butyrate alone or the combination of both activated gene expression in all the cell lines, whether or not promoter had detectable methylation (Fig. 4).

The RASSF1A gene was methylated in 3 cell lines and all three showed a complete lack of expression. None of the unmethylated cell lines showed evidence of down-regulated expression of RASSF1A mRNA. Two of three cell lines with RASSF1A methylation and complete lack of expression reactivated upon treatment with 5-Aza-2' deoxycytidine alone or in combination with n-butyrate. One of the methylated cell lines (HT-3), however, did not reactivate gene expression after treatment. The unmethylated cell lines showed normal expression and the treatment had no effect on expression.

MGMT was methylated in two cell lines. One of the two methylated cell lines (CaSki) showed a complete absence of expression while the other methylated cell line (SW756) showed normal levels of expression. Treatment with demethylating and deacetylating inhibitor did not activate the expression in any cell line regardless of promoter methylation. MLH1 gene was methylated in one cell line (SW756) but we found no evidence of down-regulated expression in this cell line (data not shown).

A total of 82 tumor biopsies derived from previously untreated primary invasive CCs and eight cell lines were used. The tumor biopsies were ascertained from patients evaluated at the Instituto Nacional de Cancerologia (Santa Fe de Bogota, Colombia) and from the Department of the Obstetrics and Gynecology of Friedrich Schiller University, Jena, Germany after appropriate informed consent and approval of protocols by institutional review boards. The primary tumors were clinically classified as FIGO stage IB (17 tumors), IIB (19 tumors), IIIB (43 tumors), and IV (3 tumors). Histologically, 77 tumors were classified as squamous cell carcinoma (SCC) and five as adenocarcinoma (AC). Clinical information such as age, stage and size of the tumor, follow-up after treatment was collected for all patients. Patient follow-up data was collected from the review of institutional medical records, and by contacting outside physicians and institutions. The follow up ranged from 1–72 months. Thirty-nine cervical intraepithelial neoplastic lesions derived from 37 patients were also obtained as formalin-fixed and paraffin-embedded archival tissues from Columbia University and Instituto Nacional de Cancerologia (Santa Fe de Bogota, Colombia). Eight normal cervical swabs, diagnosed on Pap smear as normal squamous epithelium, were collected in phosphate buffer saline from the patients attending Gynecology Clinic at the Columbia Presbyterian Medical Center, New York after appropriate informed consent. Cell pellets obtained from the swabs were used to isolate DNA. CC cell lines SiHa, SW756, C-4I, Ca Ski, C-33A, HT-3, MS751 and ME-180 were obtained from the American Type Culture Collection (Manassas, VA), and grown in tissue culture according to the supplier's recommendations. Frozen tumor tissues or cell pellets were utilized for isolating DNA and/or RNA by standard methods.

Genomic DNA was treated with sodium bisulphite as previously described 46. Placental DNA treated in vitro with SssI methyltransferase (New England Biolabs, Beverly, MA) and similarly treated normal lymphocyte DNA were used as controls for methylated and unmethylated templates, respectively. The primers used for amplification of methylated and unmethylated promoters of genes RARB, TIMP3, CDKN2A, p14^ARF, MGMT, DAPK, CDH1, GSTP1, APC promoter 1A, RB1, MLH1, TP73, BRCA1, FHIT, and HIC1 have been described previously http://pathology2.jhu.edu/pancreas/prim0425.htm#MSP; 32474849. Methylated (MF and MR) and unmethylated (UF and UR) primers for RASSF1A promoter were designed according to Herman et al 46 and as described earlier 50. PCR products were run on 2% agarose gels and visualized after ethidium bromide staining. Purified MSP products were sequenced in representative specimens by direct sequencing to confirm the methylation identified on agarose gels.

Cell lines were treated with demethylating agent 5-Aza-2' deoxycytidine (Sigma) for five days at a concentration of 2 to 5 μM, HDAC-inhibiting agent n-butyrate at a final concentration of 5 μM for the last 24 hours or a combination of both. Total RNA isolated from treated and untreated cell lines, and the total RNA and poly A⁺ RNA from normal cervix obtained from Ambion (Austin, TX) was reverse transcribed using random primers and the Pro-STAR first strand RT-PCR kit (Stratagene, La Jolla, CA). A semi-quantitative analysis of gene expression was performed in replicate experiments using 26–28 cycles of multiplex RT-PCR with β-actin (ACTB) as control and gene specific primers spanning at least 2 exons. The primers used for CDH1, MGMT, MLH1, and RARB have been described earlier 50. The gene primers used for DAPK and RASSF1A and their positions in respective cDNAs were:

DAPK-F 5'-CAGTTTGCGGTTGTGAAGAA-3' (403–422 bp)

DAPK-R 5'-CCTGCAACGAGTTCCAAGAT-3' (610–629 bp)

RASSF1A-F 5'- GACCTCTGTGGCGACTTCAT-3' (232–251 bp, exon 1A)

RASSF1A-R 5'-GCTGTTGATCTGGGCATTGT-3' (461–480 bp)

The PCR products were run on 1.5% agarose gels, visualized by ethidium bromide staining and quantitated using the Kodak Digital Image Analysis System (Kodak, New Haven, CT).

High-molecular weight DNA from frozen tumor and the corresponding peripheral blood specimens was isolated as previously described 14. A panel of 49 sequence tagged repeat polymorphic (STRP) (24 markers mapped to chromosome 2q, nine on 5p, 11 on 6p, and five on 11q) markers, previously analyzed for LOH, was chosen for scoring MSI 14151617. The criterion for analysis and classification of MSI has been described earlier 5152.

PCR amplification with PGMY09/11 L1 consensus HPV L1 primers followed by reverse line blot hybridization was used to detect 38 genital HPV types as described earlier 5354. Appropriate control experiments were set up using bulk master mix components to eliminate potential contamination. Individual assay sensitivity was analyzed by the use of serial dilutions of SiHa cell line crude cell digest, targeting 10⁴, 10³, 10², 10¹, and 10⁰ input copies of HPV-16. A no template negative control was set up in all experiments.

Four μ-thick sections were cut from paraffin-embedded blocks and immuno-stained following deparaffinization and antigen retrieval using citrate buffer at pH 6.0. Following incubation with antibodies against E-cadherin (Dako, Carpinteria, CA) and RARB (NeoMarkers, Fremont, CA), signal was detected with the Envision plus (DAKO, Carpenteria, CA) system, using diaminobenzidine as a chromogen. Tumors were considered positive for E-cadherin when cells showed membrane staining. For RARB, staining in normal epithelium either nuclear or cytoplasmic or both were considered positive. Normal epithelium and mesenchymal cell expression were used as internal controls.

We first assessed associations between presence of promoter methylation for each gene and various clinical, demographic, and genetic characteristics using Chi-Square test 55 and Fisher's Exact test (for small cell counts) 56. Characteristics that we examined included: histology, stage, tumor size, patient's age, clinical outcome, treatment response, HPV type, and microsatellite instability. Cox Proportional Hazard Models 57 were further applied to assess the relative hazard (risk) of dying associated with each methylated gene after adjusting for age, treatment status, tumor size, stage, and HPV type.