Evaluation of folate receptor 1 (FOLR1) mRNA expression, its specific promoter methylation and global DNA hypomethylation in type I and type II ovarian cancers

Two hundred fifty four ovarian cancer samples were retrospectively analysed from 1989 to 2010 at the Medical University of Innsbruck. The study was performed in accordance with the principles of the Helsinki Declaration after approval by the local ethics committee.

Median age was 61.58 years. Median follow-up was 55 months (1-289 m). After surgery patients mostly received chemotherapy as a standard treatment. A minority of them (12 pts) underwent neoadjuvant treatment (at least three courses of chemotherapy followed by interval debulking surgery); these patients were excluded from the platinum response risk analysis. The majority received adjuvant chemotherapy platinum-based therapy. Staging was performed according to the International Federation of Gynecology and Obstetrics (FIGO) classification. All FIGO stages and all histotypes were included. The clinico-pathological characteristics are reported in Table 1. According to the dualistic model proposed by Kurman et al. we divided the cancers cohort into type I and type II [24, 25]. Clinical, pathological and follow-up data were stored in a database in accordance with hospital privacy rules. Tumour samples and clinical data were collected after written informed consent of patients. After primary treatment, all patients were monitored by our department at intervals increasing from three months to one year until death or the end of the study. Follow-up information was available for all patients. Overall survival was defined as the time from diagnosis to last follow-up or death and progression-free survival as the time from diagnosis to first recurrence. Median overall survival (OS) was 55.00 months (Q₂₅-Q₇₅ 22.00-96.25), and median progression-free survival (PFS) was 25.00 months (Q₂₅-Q₇₅ 10.00-74.25). To define sensitivity to platinum we calculated the time from the last course of chemotherapy as defined by Markman et al. [26]. Moreover, we analysed tissues from 60 healthy controls (23 tubes with fimbriae and 36 normal ovarian epithelial tissues) and from 13 ovarian borderline tumours. Median age of patients with borderline tumours was 54 years and 51 years for controls; we did not find any general association between age and FOLR1 mRNA levels (r_s = 0.044, p = 0.485). In borderline tumors there were nine patients with serous and four patients with mucinous tumours. Nine patients had FIGO stage I and four patients FIGO stage II borderline disease.

Tumour specimens were obtained immediately after surgery and brought to our pathologist. Part of the tissue was pulverized under cooling with liquid nitrogen and stored at -80 °C. Total cellular RNA extraction and reverse transcription of RNA were performed as recently described [27].

Primers and probes for the TATA box-binding protein (TBP; a component of the DNA-binding protein complex TFIID as an endogenous RNA control) were used as described by Bieche et al. [28]. Primers and probes for FOLR1 were determined using the computer program Primer Express (Life Technologies, Carlsbad, CA, USA). BLASTN searches were conducted to confirm the total gene specificity of the nucleotide sequences chosen for the primers and probes. To prevent amplification of contaminating genomic DNA, the probe was placed at the junction between two exons. FOLR1 forward primer: 5′-CTG GCT GGT GTT GGT AGA ACA G -3′; FOLR1 reverse-primer: 5′- AGG CCC CGA GGA CAA GTT-3′; FOLR1 TaqMan probe: 5′-CAT TCT TCC TCC AGG GTC GAC ACT GCT-3′-BHQ1.

PCR reactions were performed using an ABI Prism 7900HT Detection System (Applied Biosystems, Foster City, CA, USA) as recently described [27].

Genomic DNA from lyophilized, quick-frozen ovarian cancer specimens was isolated using the DNeasy tissue kit (Qiagen, Hilden, Germany). Bisulfite modification was performed using the EZ DNA Methylation-Gold Kit (Zymo Research, Orange, CA, USA) according to the manufacturer’s instructions. MethyLight analysis was done as described previously [29]. The PMR value (percentage of fully methylated reference) was calculated to determine the DNA methylation measurement.

Primers and probes for COL2A1 were described recently [29]. Primers and probes for FOLR1 were determined with the computer program Primer Express (Life Technologies, Carlsbad, CA, USA) to produce a 74-base-pair PCR amplicon (nucleotide positions 17662066-17662139 as defined by NCBI Reference Sequence NT_167190.2; gi|568815271) with a mean distance of -2.384 base pairs, to the transcription start site. The CpG island in the analysed gene was identified using the CpG island searcher (http://​www.​uscnorris.​com/​cpgislands/​cpg.​cgi) that screens for CpG islands that meet the criteria and algorithm described by Daiya Takai and Peter A. Jones [30]. The following primers were used for MethyLight PCR: FOLR1: Forward: 5′-CTC GAT CTC CTA ACC TCG TAA TCC-3′; Reverse: 5′-TAT GGT GGT TCG CGT TTG TAA TT-3′, TQM-probe: 6-FAM- 5′- CCC GCC TCG ACC TCC CAA AAT ACT T-3′-BHQ1;

LINE1 DNA-hypomethylation was analysed as recently described previously [29]; the levels of unmethylated repetitive elements were expressed as percent of unmethylated reference (PUMR) values and were calculated similarly to PMR (percentage of methylated reference) values except that bisulfite-converted human sperm DNA was used as an unmethylated reference for PUR determinations.