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Supplementary Methods DNA Isolation Samples were isolated using three different methods depending on the platform used to perform the sequencing of TP53. The technique used approximately 10mg of snap-frozen tumor sample that was minced and resuspended in PBS. The sample then underwent overnight digestion with proteinase K at 55°C. DNA was isolated using the QIAmp® DNA mini Kit (Qiagen®, Maryland, USA), following the manufacturer’s protocol. Eluted DNA was quantified using the Quant-ItTM PicoGreen® system (Invitrogen®, Carlsbad, CA, USA). The second protocol also used fresh-frozen surgically resected nonrecurrent tumor and matched nonmalignant adjacent tissue that were obtained from consented patients treated for HNSCC at The University of Texas M.D. Anderson Cancer Center, under an Institutional Review Board approved protocol. Frozen tissue was embedded in optimal cutting temperature compound and cryosections from the top and middle of specimens were stained with hematoxylin and eosin prior to being evaluated by a pathologist for the presence of > 60% tumor nuclei content or absence of tumor (i.e., normal). Samples that passed this criterion were completely sectioned and washed once in PBS prior to isolating genomic DNA using an ArchivePure DNA purification kit (5Prime). The last technique isolated DNA from three slides of formalin-fixed, paraffin-embedded tissue with a region of tumor of at least 5mM x 5 mM, 10uM thickness, and at least 50% tumor density. A representative hematoxylin and eosin slide for each specimen was used to macrodissect the tumor from surrounding normal tissue. Isolated tumor samples were placed into lysis buffer and genomic DNA was isolated following the FF-PET Specimen Extraction protocol for the p53 Amplichip® (Roche Molecular Systems, Inc, Pleasanton, CA, USA). TP53 Sequencing As mentioned above three different sequencing technique were utilized to determine TP53 sequence. In each assay, the coding regions and surrounding splice sites from exons 2-11 of the TP53 gene were evaluated via direct sequencing from genomic DNA. The first sequencing protocol used was standard Sanger sequencing using BigDye® Terminator chemistry (Applied Biosystems® Life Technologies®, Carlsbad, CA, USA) and was performed through the service provided by Beckman Coulter Genomics (Beckman Coulter®, Brea, CA, USA). The second technique used exome DNA capture with Nimblegen reagents (Nimblegen) followed by sequencing on SOLiD or Illumina machines. Lastly to sequence from FFPE, DNA isolated from FFPE samples underwent PCR amplification, fragmentation and biotin labeling of the TP53 amplicons. This DNA was then hybridized to the TP53 microarray (AmpliChip®), which then proceeded to staining, washing, and laser scanning with the instrumentation as provided and specified by the manufacturer (Roche Molecular Systems, Inc, Pleasanton, CA, USA). Calculation of the EAp53 scores The evolutionary action scores for each TP53 mutation were calculated based on a simple model of the phenotype-genotype relationship, which hypothesizes that protein evolution is a continuous and differentiable process. Accordingly, the genotype ( ) and the phenotype ( ) will be related by =f ( ), and the phenotypic impact of any mutation at residue i (evolutionary action) will be the product of two terms: the sensitivity of p53 function to residue variations (f/ i) and the magnitude of the substitution ( i). The term f/ i was measured by importance ranks of the Evolutionary Trace method [1, 2], according to which, residues that vary amongst closer homologous sequences are ranked less important than those that only vary amongst distant homologous sequences. The magnitude of the substitution ( i) was measured by ranks of amino acid substitution odds [3], however, these odds were computed for different deciles of the evolutionary gradient at the substituted position. We normalized the product to become percentile scores for p53 protein, for example, an action of 68 implied that the impact was higher than 68% of all possible amino acid substitutions in p53. Statisical Classification by EAp53 Univariate Cox proportional hazards models were used to estimate hazard ratios and their corresponding p-values for all risk factors in the training set. The optimal threshold for EAp53 to stratify patients between favorable and poor outcomes was identified using the training data set, and the p-value for the estimated hazard ratio was adjusted [4]. The threshold discretizes evolutionary action into low-risk and high-risk patients. A multivariate Cox proportional hazards model was created with all risk factors except the discretized evolutionary action factor. A rank-based procedure was used based on the Cox proportional hazards model to determine the best threshold for evolutionary action in a multivariate model[5]. A Cox proportional hazard model using all risk factors as above along with the discretized evolutionary action factor is created to estimate hazard ratios for each risk factor and their p-values. The p-value for the discretized evolutionary action factor is adjusted to account for using the data to first determine the cut point. We remove covariates from the model one by one and repeat the above procedure until a model is developed containing risk factors that have hazard ratios which are significant and the p-values for the coefficient of the factors in the final model are < 0.05. Next, the threshold established in the training dataset was applied to the validation dataset to classify TP53 mutations as either low or high risk. Using survival time, univariate Cox proportional hazards model were built for each risk factor using the validation data set to get estimates for hazards ratios and their p-values. No adjustments to the p-values were necessary. A Cox proportional hazard was also built containing all the risk factors including the EAp53 threshold determined in the test set and reducing the model by removing the covariate with the largest p-value in a stepwise manner until the final model only contained significant risk factors. We used the discretized evolutionary action data to perform log-rank tests to determine differences in time to death between low-risk and high-risk TP53 patients. The above analyses for the validation data set were reproduced with disease-free survival as an outcome and time to metastases as an outcome. Generation of the HNSCC Stable Cell Lines Cells stably expressing TP53 constructs were generated as described previously [6]. Briefly, pBabe retroviral plasmids carrying the mutant TP53 constructs were transfected into the 293FT packaging cell line at 65% confluence over 8 hours in serum-free media with Lipofectamine TM 2000 (Invitrogen, Carlsbad, CA). Forty-eight hour post-transfection, media containing the virus was then collected and centrifuged at 1,000 rpm to remove cellular debris. The accepting null UMSCC1 and PCI-13 cells grown at 70% confluence were infected with the virus-containing media supplemented with polybrene. After 1 passage, the cells stably expressing the pBabe constructs were selected with 2µg/ml of puromycin and maintained for all subsequent experiments. Expression of the p53 was detected by western blotting and all cell lines were authenticated against the parental recipient cell line via short tandem repeat (STR) analysis. Immunoblotting Cells were grown on 10-cm plates to 80% confluency washed with cold PBS and then lysed with RIPA buffer. Total protein concentration was then calculated using Bio-Rad DC Protein Assay Kit (Bio-Rad Laboratories). 30ug of protein was separated on 7.5% or 12% SDS-PAGE gels and transferred to PDVF membranes. Membranes were blocked in 5% milk and probed with primary antibodies to anti-p53 (Santa Cruz Biotechnology, sc-126), NOTCH1 (C-20, sc6014, Santa Cruz Biotechnology), p21WAF1 (OP64, Calbiochem), or beta-actin (sc81178, Santa Cruz Biotechnology), and secondary antibodies to rabbit or mouse IgG linked to HRP (#7074 & #7076, respectively, Cell Signaling Technologies) in 2.5% milk. HRP was detected using the SuperSignal West chemiluminescent system (Pierce Biotechnology). Invasion Assay BD BioCoat Matrigel Invasion Chambers were rehydrated and 25,000 cells were resuspended in serum free media and seeded into the interior of the inserts. 3T3-NIH cells were used as a control due to their ability to migrate but not invade. Uncoated control inserts were used for each cell line to determine the extent of migration (BD Biosciences, 354578). Once the cells were seeded, DMEM supplemented with 10% FBS was placed into the lower well as the chemoattractant and both invasion and migration plates were incubated for 24 h at 37˚C, 5% CO2. Following incubation noninvading or migratory cells were removed from the upper surface and the remaining cells were stained and quantified at 40X magnification with the entire membrane being assessed. Each cell line was run in triplicate. The low and high risk series are a composite of three mutations, F134C, A161S, and Y236C and four mutations, R175H, H179Y, C238F, G245D, respectively and the results represent two independent experiments. The percent invasion was calculated by dividing the mean number of invading cells through the matrigel membrane by the mean number of cells migrating through the control insert membrane and multiplying this quotient by 100. Cell Proliferation Assay Cell proliferation was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as previously described[7]. Breifly, the cells were seeded at different densities and grown in a medium containing 10% FBS in 96-well tissue culture plates. After a 24-hour attachment period, the plates were assigned to different time points to obtain linear optical density (O.D.) Cells were then incubated for 3 hours in medium containing 2% FBS and 0.25 mg/mL MTT, after which the cells were lysed in 200 µL dimethylsulfoxide (DMSO) to release the formazan. The conversion of MTT to formazan was quantified with an EL-808 96-well plate reader (BioTek Instruments, Winooski, VT) set at an absorbance of 570 nm. The OD values were then obtained and analysed to determine the % of cell viability. Orthotopic Nude Mouse Model of Oral Cavity Cancer All animal experimentation was approved by the Animal Care and Use Committee (ACUC) of the University of Texas MD Anderson Cancer Center. Our orthotopic nude mouse model of oral cavity cancer has been previously validated and described in the literature[8]. UMSCC1, PCI3, and cells expressing either, a high risk, low risk TP53 mutation, a null pBabe TP53 or wildtype TP53 were harvested from subconfluent culture by trypsinization and washed with PBS. Groups of 8-10 mice were injected into the tongues with (5X104) cells suspended in 30 µL of PBS as described previously [8]. Mice were examined twice a week for 10 weeks where tumor size and weight loss were assessed and recorded. Tongue tumors were measured with microcalipers, and tumor volume calculated as (A)(B 2)π/6, where A is the longest dimension of the tumor and B is the dimension of the tumor perpendicular to A. In order to compare the tumor growth between cell lines the mean tumor volume of each cell line was plotted for each day calculated. In an effort to compare tumor growth across different TP53 mutant constructs the area under the growth curve (AUC) was calculated for each animal and the mean AUC was calculated for each group. Mice were euthanized by CO2 asphyxiation when they lost more than 20% of their preinjection body weight. Primary tumors were resected, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). The results represent three independent experiments with the high risk and low risk mutations including four mutations, R175H, H179Y, C238F, G245D and two mutations F134C and A161S respectively. Tail Vein Model UMSCC1, PCI 13, and cells expressing either, a high risk, low risk TP53 mutation, a null pBabe TP53 or wildtype TP53 were harvested from subconfluent culture by trypsinization and washed with PBS. Groups of 8-10 mice will have lung metastasis established by injecting 1 × 106 of each HSNCC cell line suspended in 200 µL of PBS into the tail vein. The mice were examined once a week for weight loss and general health. Animals were euthanatized using carbon dioxide asphyxiation if they lost more than 20% of their preinjection body weight or became moribund. The remaining mice were sacrificed at 180 days post-cell injection. All animals were necropsied, with removal of lung. Lungs were assessed both macroscopically for gross nodules and microscopically after tissues were embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). mRNA expression arrays. Total RNA was isolated from cell lines by using Tri-reagent and hybridized to Affymetrix GeneChip Human Exon 1.0ST Arrays (Affymetrix) according to manufacturer’s. Cel files were analyzed in R (v2.12.0) with aroma.affymetrix, ClassDiscovery, ClassComparison, xtable, MASS, and rms packages. Data was background corrected with RMA and quantile normalized. Sample 1495 was an outlier in RLE, NUSE, and clustering analysis and removed from further analysis. The resulting exon-level data were converted to transcripts using Core probesets definition (HuEx-1_0-st-v2,coreR3,A20071112,EP.CDF which contains 18,708 units/transcript clusters, 284,258 groups/probesets, and 1,082,385 probes) for further analysis. Hierarchical clustering used Pearson correlation and Ward’s linkage. One-way ANOVA and Dunnett tests for pairwise comparison with pBabe were applied to entire gene list. Quantitative Reverse Transcription PCR (RT-qPCR) Analyses Validation of the mRNA expression Validation of the mRNA expression array for two TP53 target genes (p21 and Notch1) was performed by RT-qPCR. Total RNA was isolated from HNSCC cell lines using Trizol method. Reverse transcription was performed using the high capacity cDNA Reverse Transcription kit (Applied Biosystem) according to the manufacturer’s protocol. Quantitative real-time PCR was performed using the CFX96 Real-time PCR Systems (Applied Biosystems, Foster City, CA) with Power SYBR Green PCR Master Mix (Applied Biosystems), using the following primers: p21 forward 5'-CGCTAATGGCGGGCTG-3', reverse 5'-CGGTGACAAAGTCGAAGTTCC-3'; Notch1 forward: 5’-ACAACGAGGTCGGCTCCTA-3’, reverse: 5’-ACAGTTCTGGCGGTGAA-3’. The GAPDH gene was used as an internal control. Triplicate samples were examined. The expression of each target gene was normalized against GAPDH which calculated by the ΔCT method (ΔΔCT = [ΔCT of target gene]-[ ΔCT of internal control gene (GAPDH)]) and the fold change of expression and standard deviation were calculated using the comparative CT method, 2(-ΔΔCT), or √((stdev(target gene)^2+stdev(control gene)^2)) respectively. 1. 2. 3. 4. 5. 6. 7. 8. Lichtarge, O., H. Bourne, and F. 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Pickering, C.R., et al., Integrative Genomic Characterization of Oral Squamous Cell Carcinoma Identifies Frequent Somatic Drivers. Cancer Discov, 2013. Sano, D., et al., Targeted molecular therapy of head and neck squamous cell carcinoma with the tyrosine kinase inhibitor vandetanib in a mouse model. Head Neck, 2011. 33(3): p. 349-58. Sano, D. and J.N. Myers, Xenograft models of head and neck cancers. Head Neck Oncol, 2009. 1: p. 32.