Evidence of a Tumour Suppressor Function for DLEC1 in Human Breast Cancer

Materials and Methods

Patients and samples. Institutional guidelines, including ethical approval and informed consent, were followed. Breast cancer tissues (n=120) and adjacent non cancerous tissues (ANCT) (n=33) were collected immediately after excision during surgery, and stored at −80°C until use. A consultant pathologist examined hematoxylin- and eosin-stained frozen sections to verify the presence of tumour cells in the collected samples. ANCT was derived from the background breast parenchyma of breast cancer patients within the study group. All tissues were randomly numbered and the details were only made known after all analyses were completed. All patients were treated according to local algorithms of management following a multidisciplinary discussion. Patients treated with breast-conserving surgery received adjuvant radiotherapy. Those with hormone-sensitive malignancy received tamoxifen. Fit patients with node-positive breast cancer or hormone-insensitive large and/or high-grade cancer were offered adjuvant chemotherapy. Medical notes and histology reports were used to extract clinicopathological data (Table I). A customised database was established to record data.

Materials. RNA extraction kits and reverse transcription kits were obtained from Sigma-Aldrich Ltd (Poole, Dorset, England, UK). PCR primers were designed using Beacon Designer (Palo Alto, CA, U.S.A.) and synthesised by Sigma-Aldrich. A custom made hot-start Master-mix for quantitative PCR was obtained from Abgene (Surrey, England, UK) (14, 15).

Tissue processing, RNA extraction and cDNA synthesis. Frozen sections of tissue were cut to a thickness of 5-10 mm and kept for routine histological analysis. Additional 15-20 sections were mixed and homogenised using a hand-held homogeniser in ice-cold RNA extraction solution. The concentration of RNA was determined using UV spectrophotometry. Reverse transcription was carried out using a reverse transcription kit with an anchored olig (dT) primer supplied by Abgene, using 1 mg of total RNA in a 96-well plate.

Quantitative analysis. The level of DLEC1 transcripts from the above prepared DNA were determined using real-time quantitative PCR based on the Amplifluor technology (15), modified from a method reported previously (15). PCR primers were designed using Beacon Designer software (15), but to the reverse primer an additional sequence, known as the Z sequence which is complementary to the universal Z probe (Intergen Inc., Oxford, UK) was added. The product expanded one intron. The primers used for each DLEC1 are detailed in Figure 1. The reaction was carried out using the following: Hotstart Q-master mix (Abgene), 10 pmol of specific forward primer, 1 pmol reverse primer which had the Z sequence, 10 pmol of FAM-tagged probe (Intergen Inc.), and cDNA from 50 ng of RNA. The reaction was carried out using the IcyclerIQ (Bio-Rad Ltd, Hemel Heamstead, England, UK), which is equipped with an optical unit that allows real-time detection of 96 reactions, under the following conditions: 94°C for 12 min and 50 cycles of 94°C for 15 s, 55°C for 40 s, and 72°C for 20 s. The levels of the transcript were generated from a standard that was simultaneously amplified with the samples. Levels of DLEC1 expression were then normalised against CK19 expression, already measured in these specimens, to correct for varying amounts of epithelial tissue between samples. CK19 transcripts were quantified as previously reported (16), using the primers detailed in Figure 1. With every PCR run, a negative control without a template was included and a known cDNA reference sample was used as a positive control.

Statistical analysis. The Mann–Whitney U-test was used for statistical analysis. DLEC1 transcript levels within breast cancer specimens were compared to ANCT and analysed against conventional pathological parameters and clinical outcome over a 10-year follow-up period. In each case, the true copy number was used for statistical analysis and hence samples were not classified as positive or negative. Statistical analysis was carried out using Minitab v.14.1 (Minitab Inc., State College, PA, USA) using a custom written macro (Stat 2005.mtw).