ABSTRACT: We searched the head and neck pathology database at The University of Texas MD Anderson Cancer Center (Houston, Texas) to identify all patients with SGCs who had been treated surgically from 1995 to 2008. Twenty cases from each tumor type, ACCs, MECs, and SDCs were selected based on the availability of at least 1.0 gm of fresh frozen tumor tissues. Seventeen salivary gland tissues (10 from matching tumor cases and 7 from neck resections from patients with other diseases) were used as control (Supplemental Table 1). The 20 ACCs had previously been included in our earlier comparative genomic hybridization analysis. 32 The tissues had been harvested immediately after resection by experienced head and neck pathologists, placed in liquid nitrogen, and stored at -80°C until use. Frozen sections of each tissue specimen were evaluated for tumor content and quality. Specimens with more than 20% of host non-neoplastic elements in any given sample underwent macrodissection to enrich the tumor percentage to more than 80%. One ACC tumor was disqualified because of excessive stroma; of the 59 tumor specimens, 14 had undergone mass dissection to remove non-tumor elements. All tissues were obtained according to an institutional review board-approved protocol for non-mucosal head and neck cancer. DNA extraction: Fresh-frozen tissue was processed using the Gentra Puregene tissue kit (QIAGEN, Valencia, CA). In brief, normal and tumor tissues were carefully dissected, and proteinase K was immediately added for cell digestion. After protein precipitation, DNA was precipitated using isopropanol, followed by a 70% ethanol washing procedure. Purified DNA was dissolved and eluted in DNA hydration solution and stored at -80°C. The quality of DNA was assessed using electrophoresis and nano-drop measurement. A 260/280 wavelength ratio of at least 1.8 was used to qualify specimens for analysis. Microarray analysis: We used the Affymetrix 250k Nsp SNP array (Affymetrix, Inc., Santa Clara, CA) to survey the genotype and DNA CNA copy number data for 59 tumor specimens and 17 healthy salivary gland specimens. Each array contains 261,563 SNP sites, which are approximately uniformly distributed throughout the genome. The SNP site mapping information was provided by Affymetrix, Inc., using human genome sequence version NCBI36/hg18. Two samples were repeatedly sampled to assess data reproducibility. Affymetrix SNP arrays were performed according to the manufacturer's directions on DNA extracted from fresh frozen tissues. Copy number analysis of Affymetrix 250k NSP arrays was performed for 76 samples, 19 ACC samples, 20 SDCs, 20 MECs and 17 controls.
