Frozen Tumor Specimens, Cell Lines, and DNA Extraction
Patient NSCLC specimens were obtained from surgical specimens at both the University of Texas M. D. Anderson Cancer Center (42 matched tumor/normal samples, 7 unpaired tumor samples) as well as from the University of North Carolina Lineberger Comprehensive Cancer Center tumor bank of surgical specimens (56 unpaired tumor samples). In both institutions, informed consent was obtained prior to surgery for the use of specimens as part of an IRB-approved protocol, in accord with the Helsinki Declaration. Tissue was snap-frozen and used for later DNA extraction. Genomic DNA was extracted from the DNA-protein phase of TriZol-extracted tissues according to the manufacturer's suggestions (Invitrogen). DNA was extracted using the PureGene kit (Gentra) on cell pellets from four HNSCC cell lines (SCC-4, SCC-9, SCC-15 and SCC-25), five lung cancer cell lines (H1395, H520, H2170, SK-MES-1 and SW-900), one breast cancer cell line (MCF7), one cervical cancer cell line (HeLa), two brain cancer cell lines (SK-N-AS and M059K), one uterine cancer cell line (AN3CA), one sarcoma cell line (HT1080), one kidney cancer cell line (HEK293), and six colon cancer cell lines (LoVo, SW48, HCT-15, DLD-1, COLO 320DM and RKO) according to the manufacturer's suggestions. All cell lines are available from ATCC (Manassas, VA). Four normal pools, each comprised of DNA from peripheral blood mononuclear cells (PBMCs) of six individuals were generated representing different genders and ages (females ≤40 yrs of age, females age >40 yrs, males ≤40 yrs and males ≥40 yrs).
TCF21 Promoter Methylation
PCR and sequencing primers were designed using the PSQ Assay Design software (Qiagen). PCR was performed in a 25 μl reactions containing Qiagen HotStart Taq master mix (Qiagen) using 1 μl bisulfate-converted DNA (about 10 ng/μl). Bisulfite conversion of genomic DNA was performed as previously reported.14
Briefly, 0.5-1.0 μg of genomic DNA was treated using the EZ-96 DNA Methylation Gold Kit (Zymo Research), including DNA sulfonation, deamination, desalting, desulfonation and recovery. Bisulfite-treated DNA was stored at −80°C until use. To reduce the cost per assay, an amplification protocol was developed using a biotinylated universal primer approach.14
Final primer concentrations were 10 nM of the reverse primer tailed with the universal primer (5′-GACGGGACACCGCTGATCGTTTA
CCAAAAAAAACCCCCTAA-3′), 100 nM of the untailed forward primer (5′-GGTAGGGTGGTTTTGAGTT-3′), and 90 nM of the universal biotinylated primer (5′-GGGACACCGCTGATCGTTTA
-3′) in each reaction. The universal primer sequence is underlined. The predicted amplicon size was 153 bp. Amplification was carried out as follows: denaturation at 95°C for 5 min, followed by 50 cycles at 95°C for 30 sec, 51°C for 1 min, 72°C for 45 sec, and a final extension at 72°C for 7 min.
Following PCR amplification, Pyrosequencing was performed on a PSQ96HS system (Qiagen) according to the manufacturer's protocol including the use of single strand binding protein (PyroGold reagents). The Pyrosequencing primer was (5′-TTGAGTTTGGAGAAGG-3′). The results were analyzed using Q-CpG software (Qiagen), which calculates the methylation percentage (mC/(mC+C)) for each CpG site, allowing quantitative comparisons. The methylation index (MI) was calculated as the average value of mC/(mC+C) for all nine of the interrogated CpG sites in the assay. Genomic DNA treated with M.SssI (New England Biolabs) was used as a universally methylated positive control; the same untreated genomic DNA amplified by whole genome amplification (GenomiPhi, GE Healthcare) was used as a universally unmethylated negative control.
Decitabine Treatment and Quantitative Real-Time RT-PCR
Three colon cancer cell lines (DLD-1, HCT-15 and RKO) with high levels (>85%) of TCF21
promoter hypermethylation were plated at a density of 500,000 cells/T75 flask. DLD-1 and HCT-15 cells were grown in RPMI-1640 supplemented with 10% FBS and 1% penicillin/streptomycin, RKO cells in EMEM supplemented with 10% FBS and 1% penicillin/streptomycin. Drug treatment with 1 μM decitabine (Sigma-Aldrich, St. Louis, MO) was started 3 hrs after seeding. Culture medium and drug were changed daily for treated and untreated cells. Cultures were grown for a minimum of four days until 80% confluency. Total cellular RNA was isolated using TRIZol reagent (Invitrogen). Input RNA (1 μg) was reverse-transcribed using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA). TCF21 expression was assessed by TaqMan qRT-PCR using assays Hs00162646_m1 and Hs01546814_m1 (Applied Biosystems, Foster City, CA, USA) covering exon 1-2 and exon 2-3, respectively. qRT-PCR was carried out as follows in 20-μl final reaction volume using 55 ng of RNA-equivalents as cDNA input: initial denaturation at 95°C for 8.5 min, followed by 45 cycles at 95°C for 15 sec and 60°C for 1 min according to the manufacturer's suggestions. GUSB (Hs99999908_m1) was used as endogenous housekeeping gene control for normalization. Each assay was performed in triplicate. Relative expression levels were calculated using the
method and scaled.
LOH and Mutation Detection
Primers were designed for detection of four microsatellites within and flanking TCF21. Primer sequences are shown in . All forward primers were 5′-tailed with 5′-GACGGGACACCGCTGATCGTTTA-3′ and all reverse primers were 5′-tailed with 5′-GTTTCTT-3′. A universal primer with the sequence 5′-GGGACACCGCTGATCGTTTA-3′ end-labeled with either FAM, HEX, or NED was used in all microsatellite amplifications. PCR conditions for the three primer reactions were as described above for amplification using the universal biotinylated primer. Amplification products were pooled as appropriate and analyzed by capillary electrophoresis on an ABI 3100 Genetic Analyzer (Applied Biosystems).
Primers for LOH and Mutation Detection in TCF21
The coding region of TCF21 (exons 1 and 2) was sequenced in both directions in four fragments. In all, 45 lung cancer samples showing zero or one hit were sequenced. Samples which had already been scored as having two hits were not sequenced. Primer sequences are shown in . All forward primers were 5′-tailed with M13 forward sequence 5-TGTAAAACGACGGCCAGT-3′, and all reverse primers with M13 reverse 5′-CAGGAAACAGCTATGACC-3′. After amplification, samples were treated with Exo-SAP (Amersham), sequenced using Big Dye Terminator v3.1 (Applied Biosystems) under standard conditions and products purified by ethanol precipitation, dehydrated in a vacuum centrifuge, and resuspended in 20 μl formamide before capillary electrophoresis on an ABI 3100 Genetic Analyzer. Sequences were aligned and visualized using Sequencher software (Gene Codes). Fragment 1 contained a polymorphic (CT)n simple tandem repeat of 8 to 12 units, which, when polymorphic, was used to confirm retention-of-heterozygosity identified by the microsatellites.
Archival NSCLC Case Selection and Tissue Microarray Construction
We obtained archival, formalin-fixed and paraffin-embedded (FFPE) material from surgically resected lung cancer specimens containing tumor and adjacent lung tissues from the Lung Cancer Specialized Program of Research Excellence (SPORE) Tissue Bank at The University of Texas M. D. Anderson Cancer Center, which was approved by the Institutional Review Board. Tumor tissue specimens from 300 NSCLCs (191 adenocarcinomas, and 109 squamous cell carcinomas) were histologically examined, classified using the 2004 World Health Organization (WHO) classification system,15
and selected for tissue microarray (TMA) construction. After histologic examination, TMAs were constructed using triplicate 1-mm diameter cores from each tumor. Detailed clinical and pathological information, including demographic data, smoking history (never- and ever-smokers) and status (never, former, and current smokers), pathologic TNM staging,16
overall survival, and time of recurrence, was available in most cases (). Patients who had smoked at least 100 cigarettes in their lifetime were defined as smokers, and smokers who quit smoking at least 12 months before lung cancer diagnosis were defined as former smokers.
Clinical Characteristics of Patient Samples on the NSCLC Tissue Microarray and Correlation with TCF21 Expression
Immunohistochemical Staining and Evaluation
An anti-human TCF21 antibody was used for immunostaining (ab32981, Abcam). FFPE tissue histology sections (5-μm thick) were deparaffinized, hydrated, heated in a steamer for 10 min with 10 mM sodium citrate (pH 6.0) for antigen retrieval. Peroxide blocking was performed with 3% H2O2 in methanol at room temperature for 15 min, followed by 10% bovine serum albumin in TBS-t for 30 min. Slides were incubated with primary antibody at 1:200 dilution for 65 minutes at room temperature. After washing with TBS-t, incubation with biotin-labelled secondary antibody for 30 min followed. Finally, samples were incubated with a 1:40 solution of streptavidin-peroxidase for 30 min. The staining was then developed with 0.05% 3′,3-diaminobenzidine tetrahydrochloride prepared in 0.05 mol/l Tris buffer at pH 7.6 containing 0.024% H2O2 and counterstained with hematoxylin. FFPE lung tissues having normal bronchial epithelia were used as positive control. For a negative control, we used the same specimens used for the positive controls, replacing the primary antibody with PBS.
TCF21 immunostaining was detected in the cytoplasm of epithelial and tumor cells. Immunohistochemical expression was quantified by microscope observation by two pathologists (M.S. and I.W.) using a four-value intensity score (0, 1+, 2+ and 3+) and the percentage of the reactivity extent. A final score was obtained by multiplying both intensity and extension values (range 0-300), and four levels of expression were arbitrarily calculated based on that score: (a) negative (score 0-9); (b) low (score 10-100); (c) intermediate (score 100 to 199); and (d) and high (score 200-300). Levels and scores were used for analysis.
EGFR Mutation Analysis
Exons 18 through 21 of EGFR
were PCR amplified using intron-based primers as previously described.17, 18
From microdissected FFPE cells, ~200 cells were used for each PCR amplification. All PCR products were directly sequenced using the PRISM dye-terminator cycle sequencing method (Applied Biosystems). All sequence variants were confirmed by independent PCR amplifications from at least two independent microdissections and DNA extraction, and sequenced in both directions, as previously reported.
The clinical and pathological data were summarized using descriptive statistics and frequency tabulations. Wilcoxon rank-sum and Kruskal-Wallis tests were used to compare biomarker expression among different prognostic factor levels. The generalized linear model was used to assess the effect of prognostic factors on TCF21 expression in the multivariable setting. Fisher's exact test was used to compare the association between categorical variables. We examined the association between overall survival (OS) and recurrence-free survival (RFS) rates and TCF21 expression in NSCLC patients with stage I or II disease, who had not undergone adjuvant chemotherapy. OS was defined as the time from surgery to death or the end of the study; RFS was defined as the time from surgery to recurrence or the end of the study. Univariate and multivariate Cox proportional hazards models were used to assess the effects of TCF21 protein expression on survivals. Two-sided p-values<0.05 were considered statistically significant. All analyses were conducted using SAS (v 9.1, Cary, NC) and S-plus (v 8.0, Seattle, WA) software.