Following a comprehensive examination of the laryngopharyngeal mucosa by an otolaryngologist and obtaining consent for enrollment in the study, true vocal fold biopsy specimens were obtained from 28 individuals according to a protocol approved by the University of Wisconsin-Madison Institutional Review Board. Six specimens were eliminated from the study because of an insufficient amount of tissue available, two were excluded as their histopathology results were outside the scope of the study (i.e., papilloma), and three were excluded because of regulatory discrepancies. Seventeen specimens were used in the final analysis. All seventeen subjects had an initial clinical diagnosis of leukoplakia or suspected malignancy (). None of these patients had undergone previous clinical evaluation or lesion biopsy. Further exclusionary criteria included any coexisting disease or lesions affecting the larynx (e.g., contact ulcers, uncontrolled reflux disease, vocal fold mass lesions), prior irradiation of the larynx, surgery in the head or neck affecting the larynx, evidence of coexisting head and neck malignancy, and non-SCCa. For each participant, the biopsy specimen was sectioned for histopathologic evaluation, and the remaining tissue was reserved for later genetic analysis. Group membership was determined by the histopathology results and included subjects with nondysplastic keratotic epithelium (ND) (n = 7), dysplastic keratotic epithelium (DYS) (n = 3), and invasive carcinoma (CA) (n = 7). A medical chart review was completed before manuscript submission to identify any patients in the ND and DYS groups that developed cancer in the follow-up period. The follow-up period ranged from 1 month to 3 years.
All specimens were collected at University of Wisconsin-Madison Hospital and Clinics by faculty and residents in the Division of Otolaryngology–Head and Neck Surgery. Tissue was placed in RNAlater solution (Ambion, Austin, TX), flash frozen in liquid nitrogen, and stored at −80°C until processing. Tissue disruption and homogenization was completed using a motorized mortar and pestle (Pellet Pestle; Kontes, Vineland, NJ). An RNeasy Mini kit (Qiagen, Valencia, CA) was used to extract total RNA from all samples. The optional on-column DNase digestion using the RNase-Free DNase Set (Qiagen, Valencia, CA) was performed. RNA concentration and quantity was assessed using a Nanodrop 1000 Spectrophotometer (Thermo Scientific, Barrington, IL).
Human Cancer PathwayFinder
Three hundred nanograms of RNA from each sample were reverse-transcribed using an RT2 First Strand Kit (SABiosciences, Valencia, CA). The cDNA from each sample was added to the RT2 SYBR Green/ROX qPCR Master Mix and aliquoted individually into a 96-well RT Profiler PCR Array-Human Cancer PathwayFinder (SABiosciences, Valencia, CA) according to manufacturer instructions. This assay profiles the expression of 84 genes involved in six tumorigenesis pathways (adhesion, angiogenesis, cell cycle control and DNA damage repair, apoptosis and cell senescence, signal transduction molecules and transcription factors, and invasion and metastasis). RT-PCR was completed in a 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA). Amplification was performed under the following conditions: 95°C for 10 minutes, 40 cycles of 95°C for 15 seconds, 60°C for 1 minute, immediately followed by a melt curve of 95°C for 15 seconds, 60°C for 1 minute, and 95°C for 30 seconds. Relative quantitative analysis was performed. RPL13A was selected as the housekeeping gene, as raw cycle threshold (Ct) values across the samples were consistent. To obtain normalized Ct values for each gene (ΔCt), the raw Ct value of the housekeeping gene (RPL13A) was subtracted from the raw Ct value of the gene of interest. Average ΔCt values for each gene were obtained by pooling the normalized values from each sample in the group. For group comparisons, the difference between the average ΔCt value between groups was calculated (ΔΔCt), and the fold change was determined using the formula: 2−ΔΔCt.
Validation of our Human Cancer PathwayFinder array results was performed with RT-PCR for four experimental genes and one housekeeping gene using the 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA). The specificity of primers was assessed using PCR, which yielded a single DNA band of expected size on agarose gel () (Integrated DNA Technologies, Coralville, IA).
Primers Used for Real-Time Polymerase Chain Reaction Validation.
One-way analysis of variance was used to determine whether the average ΔCt values were different between the groups. Pairwise comparisons were performed using Fisher protected least significant difference tests. P < .02 was considered significant. As we were using this as a screening tool to select genes for further study, no corrections were made for multiple comparisons. Results were obtained using SAS statistical software version 9.1 (SAS Institute, Inc., Cary, NC).