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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.
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