Evolutionary Action Score of TP53 Coding Variants Is ... · biomarker of response to cisplatin in...

Clinical Studies

Evolutionary Action Score of TP53 CodingVariants Is Predictive of Platinum Response inHead and Neck Cancer PatientsAbdullah A. Osman1, David M. Neskey2, Panagiotis Katsonis3, Ameeta A. Patel1,Alexandra M.Ward1, Teng-Kuei Hsu3, Stephanie C. Hicks4, Thomas O. McDonald5,Thomas J. Ow6, Marcus Ortega Alves7, Curtis R. Pickering1, Heath D. Skinner8, Mei Zhao1,Eric M. Sturgis9, Merrill S. Kies9, Adel El-Naggar10, Federica Perrone11, Lisa Licitra12,Paolo Bossi12, Marek Kimmel5, Mitchell J. Frederick1, Olivier Lichtarge3, andJeffrey N. Myers1

Abstract

TP53 is the most frequently altered gene in head and necksquamous cell carcinoma (HNSCC), with mutations occurring inover two thirds of cases; however, the predictive response of thesemutations to cisplatin-based therapy remains elusive. In thecurrent study, we evaluate the ability of the Evolutionary Actionscore of TP53-coding variants (EAp53) to predict the impact ofTP53 mutations on response to chemotherapy. The EAp53approach clearly identifies a subset of high-risk TP53 mutations

associated with decreased sensitivity to cisplatin both in vitroand in vivo in preclinical models of HNSCC. Furthermore,EAp53 can predict response to treatment and, more important-ly, a survival benefit for a subset of head and neck cancerpatients treated with platinum-based therapy. Prospective eval-uation of this novel scoring system should enable more precisetreatment selection for patients with HNSCC. Cancer Res; 75(7);1205–15. �2015 AACR.

IntroductionHead and neck squamous cell carcinoma (HNSCC) has an

incidence of over 40,000 new cases annually in the United States,

and over 500,000 worldwide with an associated disease-specificmortality exceeding 50% (1). The treatment of locally advancedhead and neck cancer has evolved over the past three decades andoften requires complex, multimodality therapy, including surgi-cal resection, and/or external beam radiation with or withoutneoadjuvant, concurrent, or adjuvant cisplatin-based chemother-apy (2, 3). Currently, there are no molecular biomarkers to guideselection among these various treatment options. TP53 is themostfrequently altered gene in human cancers and recent data fromwhole-exome sequencing of HNSCC reveals that this gene ismutated in 60%–80%of humanpapillomavirus negative (HPV�)cases (4, 5). The TP53 gene has been called the "cellular gate-keeper" due to its central role in response to cell stressors such asDNA damage, hypoxia, and oncogenic stress. Cellular DNAdamage often leads to stabilization and accumulation of wtp53,which in turn leads to enhanced transcription of p21 and subse-quently cell-cycle arrest, apoptosis, and senescence. The increasein p53 stability depends critically on the phosphorylation ofserine/threonine residues (6–9).

Although mutations in TP53 have been shown to have predic-tive significance for response to platinum-based therapy in severalstudies, it remains unclear how to stratify patients into responsecategories based on TP53 status (10–14). Recently, we developedan algorithm termed Evolutionary Action (EAp53) that accuratelystratifies patients whose tumors have TP53 mutations associatedwith especially poor outcomes (high risk), from other mutationswith outcomes similar to patients with wild-type TP53 (low risk)and have validated EAp53 as a reliable prognostic marker (D.M.Neskey and colleagues; submitted for publication). We hypoth-esize that high-risk p53mutations identified by the EAp53 scoringsystem are associated with an abnormal functional activity that

1Department of Head and Neck Surgery, The University of Texas MDAnderson Cancer Center, Houston, Texas. 2Department of Otolaryn-gology Head and Neck Surgery, Hollings Cancer Center, MedicalUniversity of SouthCarolina,Charleston, SouthCarolina. 3Departmentof Human and Molecular Genetics, Baylor College of Medicine, Hous-ton, Texas. 4Department of Biostatistics and Computational Biology,Dana-FarberCancer Institute, Boston,Massachusetts. 5DepartmentofStatistics, Rice University, Houston, Texas. 6Department of Otolaryn-gology Head and Neck Surgery, Albert Einstein School of Medicine,Bronx, New York. 7Department of Internal Medicine, Tufts MedicalCenter, Boston, Massachusetts. 8Department of Thoracic RadiationOncology,The University of Texas MDAnderson Cancer Center, Hous-ton, Texas. 9Department of Thoracic/Head and Neck Medical Oncol-ogy, The University of Texas MD Anderson Cancer Center, Houston,Texas. 10Department of Pathology,The University of Texas MDAnder-son Cancer Center, Houston, Texas. 11Department of Pathology, Fon-dazione Istituto Di Ricovero e Cura a Carattere Scientifico, IstitutoNazionaleTumori,Milan, Italy. 12HeadandNeckMedicalOncologyUnit,Fondazione Istituto Di Ricovero e Cura a Carattere Scientifico, IstitutoNazionale Tumori, Milan, Italy.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

A.A. Osman and D.M. Neskey contributed equally to this article.

Corresponding Author: Jeffrey N. Myers, Department of Head and NeckSurgery, The University of Texas MD Anderson Cancer Center, 1515 HolcombeBlvd, Houston, TX 77030-4009. Phone: 713-745-2667; Fax: 713-794-4662;E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-14-2729

�2015 American Association for Cancer Research.

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contributes to cisplatin resistance in head and neck cancer. There-fore, to determine whether EAp53 has utility as a predictivebiomarker of response to cisplatin in HNSCC, we used bothpreclinical laboratory-based models and retrospective clinicaldata to assess the response of tumors expressing no p53, wtp53,or a series of low- and high-risk p53 mutations to cisplatin. Thefirst aspect of our preclinical model found that in clonogenicsurvival assays, cell lines expressing high-risk p53 mutations aremore resistant to cisplatin treatment than cell lines expressinglow-risk mutations or wild-type p53.

To further characterize the preclinical response of the TP53mutations stratified by Evolutionary Action to cisplatin thera-py, tumors harboring these mutations were created in anorthotopic mouse model of tongue cancer. Concordant withthe differential effect of TP53 mutations on cisplatin responseobserved in vitro, mice with tumors harboring wild-type p53 orlow-risk mutations showed a significant response to cisplatintherapy, whereas the tumors derived from cells either null forp53 expression or with high-risk p53 mutations did not showany growth inhibition with cisplatin therapy. In an effort tocorrelate the clinical utility of the EAp53 to predict response tocisplatin in patients with HNSCC, the TP53 mutational statusof a patient cohort of 68 patients treated for locally advancedHNSCC of the oral cavity with cisplatin-based induction che-motherapy followed by surgical resection was determined.Results from this analysis confirmed our preclinical findingswherein patients' tumors with high-risk mutations were signif-icantly less responsive to cisplatin-based chemotherapy thantumors with low-risk mutant or wild-type p53. These resultsindicate that the TP53 mutational status may be a usefulbiomarker for predicting response to cisplatin-based chemo-therapy in HNSCC patients.

In summary, our data clearly demonstrate that high-risk TP53mutations are associated with decreased sensitivity to cisplatinnot only in preclinical studies but also in an analysis of aneoadjuvant chemotherapy clinical trial. Prospective clinicalstudies will be necessary to confirm the utility of TP53 statusstratified by EA as a predictive biomarker of response to cisplatin-based therapy for HNSCC patients, which will potentially enablethe personalization of therapy for patients that will most likelybenefit from this treatment strategy.

Materials and MethodsCell lines

Two HNSCC cell lines, UMSCC-1 and PCI-13, were selectedfor their lack of p53 expression due to a splice-site in UMSCC1(hg19:chr17:7578370C>T) and a deletion in PCI13 (hg19:chr17:7579670_7579709del). UM-SCC-1 was provided by Dr.Thomas Carey (University of Michigan, Ann Arbor, MI) in Feb-ruary 2010. PCI-13 was acquired from Dr. Jennifer Grandis(University of Pittsburgh, Pittsburgh, PA) in August 2008. Thenaturally occurring HNSCC cell lines, HN30 (wtp53) and HN31(mutp53) were obtained in December 2008 from the laboratoryof Dr. John Ensley (Wayne State University, Detroit, MI). The celllines and their isogenic derivatives were tested and authenticatedagainst the parental cell lines by our group using short-tandemrepeat analysis (15) within 6 months of use for the current study.Details regarding cell culture, reagents, and generation of stablecell lines were previously described (D.M. Neskey and colleagues;submitted for publication).

Classification by evolutionary action scoring systemMissense TP53 mutations were divided into "high-risk" and

"low-risk" groups based on themodel described previously (D.M.Neskey and colleagues; submitted for publication).

ImmunoblottingCells grown on 10-cm plates were treated with clinically rele-

vant dose of cisplatin (1.5 mmol/L) for 24 hours and washed withcold PBS. Western blotting was performed using standard tech-niques previously described (16) and primary antibodies to anti-p53 (Santa Cruz Biotechnology, sc-126), anti-phospho-p53 ser-ine 15 (Cell Signaling Technology, 9284), anti-p21 (Calbiochem,OP64), and anti-b-actin (Sigma Aldrich, A1978) were used.

Transcriptional activity of TP53Transcription of p21, a canonical p53 target, was measured via

luciferase reporter activity using a vector containing the 2.4 kbp21promoter andfirefly luciferase (pWWP-LUC;Addgene).UMSCC1and PCI-13 cells expressing various TP53 constructs, HN30, andHN31 were cotransfected with pWWP-LUC and a constitutivelyactive Renilla luciferase construct using Lipofectamine 2000. After48 hours, cells were treated with 1.5 mmol/L cisplatin and incu-bated for 24 hours before collection. Luciferase reporter activitywasmeasured as previously described (17). The results for the p21reporter assay are relative to the cisplatin-treated wild-type (WT),which was standardized to 100 relative light units.

Quantitative reverse transcription PCR analysesThe effect of TP53 mutants on transcription of three down-

stream target genes (p21,MDM2, andNOXA) were determined byqRT-PCR. HNSCC (UMSCC1, PCI-13) cells stably expressing theTP53-mutant constructs were treated with cisplatin (1.5 mmol/L)for 24 hours before isolation of total RNA using RNeasy MiniKit (QIAGEN). Reverse transcription was performed using thehigh capacity cDNA Reverse Transcription Kit (Applied Biosys-tems) according to the manufacturer's protocol and a detaileddescription is included in the Supplementary Materials andMethods. The GAPDH gene was used as an internal control.Triplicate samples were examined. The expression of each targetgene was normalized against GAPDH, which was calculated bythe DCt method [DDCt ¼ DCt of target gene � DCt of internalcontrol gene (GAPDH)], and resultswere presented as fold changeof expression.

mRNA expression arraysTotal RNAwas isolated from cell lines by using TRI reagent and

hybridized to Affymetrix GeneChip Human Exon 1.0ST Arrays(Affymetrix) according to manufacturer's instructions and adetailed description is included in the Supplementary Materialsand Methods. The expression of TP53 target genes in pBabe andeach of other groups was calculated and heatmaps were generateddepicting the expression patterns of these genes.

Clonogenic survival assayHNSCC cells stably expressing the TP53 constructs were

seeded in 6-well plates at various densities, which allowed forapproximately equal number of colonies in the control wellsfor each construct. The next day, cells were treated with increas-ing doses of cisplatin (0.01–2 mmol/L) dissolved in dimethylsulfoxide (DMSO) for 24 hours and cultured for 10 to 14 days

Osman et al.

Cancer Res; 75(7) April 1, 2015 Cancer Research1206




to allow for colony formation of at least 50 cells. The cells werestained with crystal violet and analyzed as previously described(17). Each experiment was repeated more than three times andtreatments were performed in triplicates. An IC50 for each TP53construct was calculated as the mean IC50 from each clonogenicassay.

Orthotopic nude mouse model of oral cavity cancerAll animal experimentation was approved by the Animal Care

and Use Committee of the University of Texas MD AndersonCancer Center. Our orthotopic nude mouse model of oral cavitycancer has been previously validated and described in the liter-ature (18). UMSCC1, PCI 13, and cells expressing either, a high-risk, low-risk TP53 mutation, a null pBabe TP53 vector or wild-typeTP53 alongwithHN30andHN31 cellswere used in the studyand a detailed description of the technique is included in theSupplementary Materials and Methods.

Patient cohort and TP53 sequencingA cohort of 68 patients with oral cavity squamous cell carci-

noma (SCC) treated with platinum-based induction chemother-apy followed by surgery was collected from two clinical trials toinvestigate the predictive value of EAp53. Patient demographic,clinical data, and cisplatin-based treatment regimens were previ-ously published (12, 19). Patient data, specimens, and TP53sequences were collected under Institutional Review Board–approved protocols. DNA was extracted from tissue of patientsenrolled in the trials and different techniques were used todetermine TP53 sequence (20, 21). Detailed description of DNAisolation and TP53 sequencing is included in the SupplementaryMaterials and Methods. Patients with either TP53 wild-type ormissense mutations were then scored by the EAp53 system intolow- or high-risk categories as previously described (D.M. Neskeyand colleagues; submitted for publication). The EvolutionaryAction classification score was then correlated with clinicopath-ologic factors andpatient outcome to determine associationswithtreatment response and survival.

Statistical analysisANOVA analysis with Student t tests were carried out to analyze

in vitro data. For mouse studies, the two-tailed t test was used tocompare tumor volumes between control and treatment groups.Survival was determined using the Kaplan–Meier method andcomparedusing log-rank tests. Fisher exact test orc2 testwere usedto calculate the ORs between treatment and clinical response. Pvalues <0.05 were considered significant.

ResultsDNA damage-induced functional activity of p53 in response tocisplatin treatment is impaired in HNSCC cells expressing low-and high-risk TP53 mutations

To examine in preclinical models whether the response tocisplatin therapy correlates with TP53 mutational status strat-ified by the Evolutionary Action method, the p53 function ofcell lines that either exogenously express various p53 constructsincluding wild-type, low-, or high-risk mutant isoforms orendogenously express wild-type p53 (HN30) or a high-riskmutation (HN31) was assessed in these cells following treat-ment with cisplatin and analyzed by Western blot analysis. Asexpected, low basal expression levels of p53 and p21 were

increased after cisplatin treatment in cells expressing wild-typeTP53 (Fig. 1A and B and Supplementary Fig. S1A). In additionto the p53 and p21 induction after cisplatin treatment in thecell lines stably expressing wtp53, there was a similar level ofp53 phosphorylation observed in the cells compared with theHN30 cell line, which endogenously expressed wtp53, indi-cating that the stably expressed wild-type TP53 was function-ally active. In contrast, cells expressing mutant TP53 endoge-nously or exogenously, had higher basal levels of p53 withminimal induction of p53 or p21 after cisplatin treatment,indicating a lack of functional p53 (Fig. 1A and B and Sup-plementary Fig. S1A). Regardless of mutational status, phos-phorylation of p53 following cisplatin was a ubiquitous find-ing, but p21 induction was most evident in cells expressingwtp53 and low-risk mutant p53. The data suggest that whilep53 phosphorylation occurs in response to DNA damage,many of the high-risk mutations are not functional with respectto p21 induction, and that low risk mutations may retainpartial wtp53 function.

Mutations in TP53 alter the DNA binding domain confor-mation and disrupt the ability of p53 to bind to target genepromoters and consequently to transactivate downstream genes(22) Thus, the ability of TP53 mutants to modulate the expres-sion of classical wtp53 responsive target genes such as p21,MDM2, and Noxa was examined by both p21 promoter lucif-erase assay and qRT-PCR. After cisplatin treatment, cells withwtp53 have an increase in promoter activity while the low-riskmutations have a stable level of activity and high risk p53 havesuppressed levels of p21 promoter activity relative to their basallevels and to the basal levels of the empty vector control (Fig.1C and Supplementary Fig. S1B). The mRNA levels of the targetgenes, p21, MDM2, and Noxa were significantly elevated inresponse to cisplatin treatment in HNSCC cells harboring eitheran exogenously expressed or endogenous wild-type p53. Cellswith low-risk mutations in the PCI13 cell line showed a trendtoward increased target gene expression following cisplatintreatment relative to cells lacking p53, specifically the p21 levelin A161S, or MDM2 and Noxa levels in Y236C (Fig. 1D). This isin contrast to cells harboring high-risk mutations where themRNA levels after cisplatin treatment were similar to cellslacking p53 (Fig. 1D). Similar observations were seen in thelow-risk UMSCC1 cell lines, specifically MDM2 level in A161Sor MDM2 in Y236C. UMSCC1 cells harboring high-risk muta-tions were more variable in the target gene expression aftercisplatin treatment, which may represent unique properties ofthese constructs or the cell background (Supplementary Fig.S1C). The difference between p21 mRNA and protein levelsobserved in PCI13 and UMSCC1 cells expressing the high-riskmutant (R175H) is possibly due to posttranslational modifi-cation event that resulted from less degradation of the mutantprotein and therefore enhanced its stability upon cisplatintreatment. It could also be related to p63 and p73 isoformsbeing differentially expressed in these cells upon cisplatinaddition. The p63 and p73 are well known p53-related proteinsthat act as transcriptional activators of p21 and apoptotiocinducers upon DNA damage in tumor cells (23). Taken togeth-er, these results reveal that cells with a mutated p53 haveincreased basal levels of protein but following cytotoxic stress,there is decreased promoter activity of the canonical target, p21,and low mRNA levels of downstream target genes comparedwith cells expressing wild-type p53.

EAp53 Predicts Response to Cisplatin in Head and Neck Cancer

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HNSCC cells bearing high-risk TP53 mutations are highlyresistant to cisplatin treatment in vitro

To determine whether EAp53 has utility as a marker that canpredict HNSCC response to cisplatin therapy, we assessed theresponse of HNSCC cells expressing no p53 (pBabe empty vec-tor), wtp53, or a series of low- and high-risk p53 mutations tocisplatin in clonogenic survival assays. Figure 2A is representativeimages of clonogenic survival assay inHNSCCcell lines. As shownin Fig. 2B and C, in both genetic backgrounds, the high-riskmutant p53 clones were highly resistant to cisplatin, with 4 of5 clones having an average IC50 of 0.95 mmol/L > 0.8 mmol/L,when exposed to cisplatin for a 24-hour period. We have deter-mined this in vitro exposure of 0.8 mmol/L to be equivalent to thehighdose of cisplatin (i.e., 100mg/squaremeter) given to patientsbased upon pharmacokinetic area under the curves (AUC) studiesin humans. Low-risk mutant p53 clones were less resistant tocisplatin with an average IC50 of 0.72 mmol/L that is statisticallysignificant when compared with the high-risk mutant p53 clones(Fig. 2B and C). In addition, the clones expressing wild-type p53had lower IC50 values (0.15 mmol/L) compared with clones withnull pBabe empty vector (0.44 mmol/L) in both PCI-13 (P <0.001) and UMSCC1 (P < 0.003). These data suggest that intro-duction of low- and high-risk TP53 mutations into HNSCC celllines resulted in a gain-of-function (GOF) phenotype for resis-tance to cisplatin therapy. Furthermore, our laboratory has shownthat the endogenous mutp53 of HN31 also confers a relativecisplatin resistance with an IC50 of 0.60 mmol/L comparedwith itsisogenic wtp53 counterpart, HN30, which has an IC50 of 0.14mmol/L (24). To further address loss-of-function (LOF) versusGOF, TP53 was knocked down in the isogenic pair of cell linesHN30 (wtp53) and HN31 (HRmutp53) and cells were thenexamined for cisplatin sensitivity (Supplementary Fig. S2A andS2B). The shRNAp53 HN30 cells become more resistant tocisplatin (IC50: 0.32 mmol/L), arguing that loss of wtp53 functioncan make tumors less sensitive to cisplatin. Interestingly, knock-

down of mutant HR p53 in HN31 made cells more sensitive tocisplatin with an IC50 value very close to the HN30 p53 knock-down (IC50 of 0.30 mmol/L vs. 0.32 mmol/L), indicating a GOFassociated with the HRmutation. Collectively, the data argue thatin vitro both loss of wtp53 and aHR-associated GOF contribute toincreased cisplatin resistance.

Expression profile of high-risk TP53mutations shows a lack ofp53 transcriptional activity while low-risk mutations retainsome residual function

Given the apparent GOF phenotype seen in our in vitro studiesof the p53 mutants, we performed mRNA expression profiling inan effort to identify genes and pathways specific to the high-riskmutp53 that could explain their relative resistance to cisplatin.The principal component analysis of the gene expression profilesfor cisplatin-treated UMSCC1 cell lines, wtp53, pBabe, low-riskmutation (A161S), or high-risk mutation (C238F), revealed thatthe high-risk mutation expression profiles were more similar tothe pBabe cell line, which lacks p53 expression. This is apparentfrom component 1 (x-axis), which accounted for 40% of variancein expression. Furthermore, the low-risk mutation profilehad smaller variances (20%) in expression from the pBabe andhigh-risk mutations as seen by the large component 2 (y-axis)contribution. In contrast, thewtp53had the largest variances fromthe high-risk mutation and pBabe and was also distinct fromthe low-risk mutation profiles (Fig. 3A and B).

Ordinal logistic regression models were performed to identifygenes that contribute to either a GOFor LOF phenotypewhere themagnitude of expression fromhighest to lowest is either high-risk,low-risk, wtp53, and pBabe or wtp53, low-risk, high-risk, andpBabe respectively. Surprisingly, based on our in vitro data, thenumber of significant genes with the false discovery rate set at<10%, that contribute to a LOF phenotype was dramaticallyhigher than the number of genes associated with a GOF pheno-type, 1190 and 0, respectively (Fig. 3B and C). Furthermore, the

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Figure 1.Functional activity of TP53 inducedby DNA damage is impaired inHNSCC expressing low- and high-risk TP53 mutations. A and B,Western blot analysis of isogenicHNSCC cell line, PCI-13, that stablyexpresses wild-type or mutated p53constructs or HN30 and HN31HNSCC cell lines that endogenouslyexpress wild-type TP53 andmutated TP53, respectively. C, p21reporter luciferase activity of thesame PCI13 isogenic cells lines alongwith HN30 and HN31. D, qRT-PCR ofp21, MDM2, and Noxa in PCI-13isogenic cells lines and HN30 andHN31 HNSCC cell lines. CDDP,cisplatin. � , significant change,P < 0.05, from cisplatin-treatedwtp53; significant increase inactivity or expression above basallevels.

Osman et al.





beta-uniform mixture plot analyses for the two potential phe-notypes reveal an enrichment of genes, with low P values leadingto nonuniform distribution of genes in LOF analysis comparedwith the more uniform distribution in the GOF analysis (Fig. 3Dand E). These results validate the higher P value cutoff used inthe ordinal logistic regression analyses for LOF compared withGOF (Fig. 3B and C). As expected, the primary pathways thatwere driving these expression patterns were regulated by p53.Overall, the results from themRNA expression profile reveal thatthe introduction of a high-risk p53mutation leads to a reductionin the wild-type p53 function to levels similar to pBabe cellsthat lack p53 expression. In addition, introduction of a low-risk mutation results in an expression pattern suggestive ofresidual wild-type p53 function as seen by the intermediatelevel of expression of TP53 target genes (Fig. 3F and Supple-mentary Table S4).

High-risk TP53 mutations are associated with decreasedresponse to cisplatin therapy and overall survival in anorthotopic mouse model of tongue cancer

To further characterize the ability of the EAp53 to predictresponse of the mutations to cisplatin therapy, tumors harboring

thesemutations were created in an orthotopic nudemousemodelof tongue cancer. Animals underwent a 4-week course of cisplatintherapy, during which the tumor volumes and overall survivalwere monitored. Consistent with the differential effect of TP53status on cisplatin response observed in vitro, tumors in miceinjected with tumor cells expressing wild-type p53 showed asignificant response to cisplatin therapy, while the tumors derivedfrom cells null for p53 expression harboring the pBabe vectorcontrol or high-risk p53 mutations did not show any growthinhibition with cisplatin therapy (Fig. 4A–C). Interestingly, theresponse of tumors with low-risk mutations was more similar tothe response of wtp53-bearing cells, which in agreement with themRNA levels and expression array data in that the low-riskmutations appear to retain some wild-type p53 function. Tocompare relative tumor response between tumors to cisplatin,the area under the tumor growth curve was calculated for eachanimal and themean AUCwas plotted for each treated tumor andtheir corresponding control (Supplementary Fig. S3). Mice withtumors that harbor endogenous or exogenous wild-type p53 orlow-risk mutations have a significant response (P < 0.0001) tocisplatin therapy while mice harboring pBabe (null) or high-riskp53 mutations show minimal growth inhibition with cisplatin

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PCI-13 clones with different TP53 status UMSCC1 clones with different TP53 status

Figure 2.High-risk TP53 mutations are resistant to cisplatin in vitro. Cisplatin sensitivity was determined by the clonogenic survival assay in HNSCC isogenic cell lines(PCI-13 and UMSCC1) harboring wild-type and TP53-mutant constructs. A, representative images of colony formation assay in HNSCC cell lines. B, calculated IC50 ofcisplatin for individual TP53 constructs in both UMSCC1 and PCI-13 isogenic cell lines. Horizontal dashed line delineates the increase in cisplatin IC50 overan in vitro dose (0.8 mmol/L) that is equivalent to the high dose of cisplatin (i.e., 100 mg/square meter) given to patients based upon pharmacokinetic area underthe curves studies in humans. C, the average dose–response curve of cisplatin for low- and high-risk mutations and wild-type TP53. P < 0.001, low- andhigh-risk mutation versus wild-type TP53.






therapy. This gradient of response corroborates the expressionarray data and once again implies a partial wild-type p53 functionfor the low-risk mutations and lack of p53 function for the high-risk mutations. The resistance to cisplatin seen in the high-riskmutations was also associated with decreased survival in animalsbearing tumors with high-risk TP53mutations (Fig. 4D–F). Theseresults demonstrate that the Evolutionary Action method canpredict the p53 mutations that are least likely to respond toplatinum-based therapy in vivo in an orthotopic murine modelof oral cancer.

EAp53 classification predicts response to platinum-basedchemotherapy in patients with locally advanced oral cavitycancer

To determine the reliability of EAp53 to predict response totreatment in patients with oral cavity cancer, we identified acohort of patients with locally advanced oral cavity cancer who

received platinum-based induction chemotherapy in the contextof prospective clinical trials. This cohort consisted of 68 patients,of which 26 of the tumor samples (38%) hadmissensemutationsof TP53, while 42 tumor samples (62%) hadwild-type p53 (Table1). We have shown that the EAp53 system identified three groupsindependently, lowEAp53 score, high EAp53 score, andwild-typep53 (D.M. Neskey and colleagues; submitted for publication).Univariate analysis in the training set revealed that the low EAp53score mutations, that is, low risk, and wild-type were not statis-tically different, whereas the high EAp53 score mutations, termedhigh-risk mutations, appeared to be distinct from the other twogroups (D.M. Neskey and colleagues; submitted for publication).Given the similar outcomes, patients with tumors having low-riskmutations were combined with wild-type p53 (wtp53). There-fore, the TP53 status was further classified by EAp53 into low-riskgroup (42wild-type TP53 and 12 low-risk TP53mutations), and ahigh-risk group consists of 14 high-risk TP53 mutations (Table 1

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161169

5.29e−056.76e−048.65e−033.84e−02

Figure 3.Gene expression array analysis reveals that introduction of high-riskmutations results primarily in a LOF phenotype. A, principal component analysis of the variancein gene expression between cisplatin-treated HNSCC clones with different p53 status revealed that wtp53 had much larger variance in expression than pBabeandhigh-risk p53mutations (component 1). The low-risk p53mutations also hadmore variance in expression thanpBabeandhigh-risk p53mutations (component 2),whereas wtp53 and low-risk p53 mutations had less variance compared with each other, indicating that high-risk p53 mutations resemble a LOF. B andC, ordinal regression analysis for a LOF and GOF phenotypes. LOF phenotype altered gene expression in the order of WT>low risk>high risk>pBabe, whereWT hadthe largest change and high risk and pBabe were similar (B). A GOF phenotype identified genes with altered expression in the order of high risk>low risk>wild-type>pBabe (C). Results showed that at each given statistical cutoff more genes were statistically significant in the LOF order than in the GOF order. D andE, beta-uniform mixture plots of loss and gain of function ordinal regression analyses. The beta-uniform mixture plots further indicate a stronger bias towardlow P values in the LOF order. F, heatmap of 1,190 significant genes from the LOF ordinal logistic. As the false discovery rate (FDR) indicates the rate of false positivesamong the -positives, many analyses use a cutoff close or equal to 0.1 for the false discovery rate rather than the 0.05 cutoff traditionally used for P values.Therefore, a lowest statistical cutoff that corresponded to a false discovery rate of 0.1 was chosen to differentiate between the LOF and GOF phenotypes.

Osman et al.





and Supplementary Table S1). Review of the pathologic findingsrevealed that 13 of the patients (93%) with high-risk TP53mutations had residual disease, whereas only one patient showedcomplete response to cisplatin-based therapy. In contrast, 24 ofthe 54 patients (44%) with wild-type p53 and low-risk TP53mutations achieved complete pathologic response while 30patients (56%) had residual disease. These data demonstrate thatrelative to low-risk TP53, high-risk mutations are greater than 10foldmore likely to have residual disease following cisplatin-basedchemotherapy (Table 1; P¼ 0.029). EAp53 status was also foundto be better predictor of cisplatin response than the previousclassification system developed by Poeta and colleagues (11),which showed no statistically significant association with neoad-juvant response (Table 1; P ¼ 0.248) in our cohort. The otherclinicopathologic data analyzed were not associated with aresponse to cisplatin therapy (Table 1). In addition, patients withtumors havinghigh-risk TP53mutations appear to have decreasedoverall survival relative to patients with low-risk TP53 status (Fig.5A; P ¼ 0.04) in a Kaplan–Meier analysis. There was also a trendtoward decreased disease-free survival in patients with high-riskTP53 mutations but it did not reach statistical significance

(Fig. 5B; P ¼ 0.08). On univariate and multivariate analyses, thesurvival benefit of low-risk p53 status in the log rank tests was notobserved in Cox Proportional Hazard Ratio Model (Supplemen-tary Table S2 and S3). Overall, these data provide evidence thatEAp53 can predict a subset of patient with high-risk TP53 muta-tions that have a decreased response to platinum-based chemo-therapy and a poorer overall survival.

DiscussionCurrently, there are not any established molecular biomar-

kers to predict response to chemotherapeutic agents in HNSCC.Recent whole exome analysis has confirmed that TP53 is themost frequently mutated gene in HNSCC occurring in 60%–

80% of cases but the challenge remains to identify the muta-tions associated with resistance to current cytotoxic therapiesand therefore decreased survival outcomes (4, 5). In the currentstudy, we implemented a novel classification system, EAp53,which has the ability to predict response to cisplatin-basedtherapy not only in preclinical models of HNSCC but also inpatients with locally advanced oral cavity squamous cell

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Figure 4.High-risk TP53mutations are resistant to cisplatin in an orthotopic nudemouse model. A–C, HNSCC cell lines exogenously expressing PCI 13 and UMSCC1 (A and B),and endogenously HN30 and HN31 (C) expressing p53 were injected into the tongue of nude mice. D–F, Kaplan–Meier curves of the orthotopic tongue modelcreated with either cell lines HN30 or HN31 (D) or TP53 isogenic cell lines PCI-13 or UMSCC1 (E and F).






carcinoma. We utilized a previously described collection ofisogenic head and neck squamous cell carcinoma cell linesharboring a series of TP53 mutations or wild-type TP53, whichallowed us to specifically examine the impact of TP53 altera-tions on response to cisplatin, as the genetic backgrounds ofthese cell lines are otherwise identical (D.M. Neskey andcolleagues; submitted for publication). To confirm that theexogenous expression of p53 in these cells lines accuratelyrepresents the function of both wtp53 and mutant p53

(mutp53), cells endogenously expressing either wild-type ormutant p53 were also used for comparison.

The results from this study reveal that relative to wild-typeTP53, both high- and low-risk mutations show a decrease incisplatin-mediated p21 induction, a known transcriptional targetof p53 in response to cisplatin treatment. In low-riskmutant TP53cells, this diminished p21 induction appears to be associatedwitha reduction in the p21 promoter activity and an intermediate levelof mRNA expression relative to wtp53-expressing cells. In con-trast, the cell lineswith either exogenoushigh-riskTP53mutations

Table 1. ORs of response to platinum-based therapy for various clinicopathologic features

Clinical responseCharacteristic Total Yes No OR 95% CI Pa Global Pb

All patients 68 25 43Age<59 46 19 27�59 22 6 16 1.877 (0.620–5.675) 0.265 —

GenderMale 50 19 31Female 18 6 12 0.816 (0.262–2.536) 0.725 —

T stage2 21 12 93 32 9 23 3.407 (1.070–10.847) 0.0384 15 4 11 3.667 (0.874–15.384) 0.076 0.073

N stage0 39 14 251 12 5 7 0.784 (0.209–2.938) 0.7182b 8 4 4 0.56 (0.121–2.593) 0.4582c 4 2 2 0.56 (0.071–4.421) 0.5823 5 0 5 6.255 (0.322–121.429) 0.226 0.404

N stage<2b 51 19 32� 2b 17 6 11 1.089 (0.346–3.422) 0.885

TP53 statusWild-type 42 20 22Mutant 26 5 21 3.818 (1.211–12.034) 0.022

EA statusLow risk 54 24 30High risk 14 1 13 10.4 (1.269–85.233) 0.029

Poeta classificationNon-disruptivec 61 24 37Disruptive 7 1 6 3.9 (0.440–34.387) 0.248

Abbreviations: CI, confidence interval; EA, evolutionary action score.aUsed Fisher exact test to calculate P values.bIf contingency table was larger than 2 � 2, then global P value was calculated using either c2 test or Fisher exact test and P value was calculated for each 2 � 2subtable. For comparison with the EAp53 system, patient tumors were also classified as disruptive and non-disruptive according to Poeta and colleagues (11).cPatientswithwild-type TP53or silentmutationswere classified as nondisruptive; however, the associationwas still not significant evenwhen patientswithwild-typeTP53 or silent mutations were removed.

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A BFigure 5.EAp53 classification predicts response toplatinum-based chemotherapy in patient withoral cavity squamous cell carcinoma. A, log-ranktests of Kaplan–Meier survival plots for a cohortof patients treated with platinum-basedinduction chemotherapy followed by surgeryrevealed that EAp53 can predict patients withhigh-risk mutations that have a decreasedoverall survival, P ¼ 0.041. B, there was atrend toward decreased disease free survivalin patients with high-risk TP53 mutationsthat almost reached statistical significance,P ¼ 0.08.

Osman et al.





or endogenous mutp53 had decreased p21 promoter activityfollowing cisplatin treatment as previously described (25, 26).The functional activity of high TP53 mutations was partiallycorroborated by the qRT-PCR results with the high-riskmutationshaving a decreased level of target gene expression followingcisplatin treatment. It has been suggested that loss of upregulationof key p53 target genes may contribute to the GOF phenotype ofsome mutp53 (25–28). Although mutated p53 is unable to bindto sequence specific DNA of target gene promoters secondary toalterations in theDNAbinding domain, it has been suggested thatp53 either recognizes target promoters independent of this regionor binds the promoters at regions unique from p53-binding sites(26, 29). Furthermore, the loss of upregulation of p53 target genesmay be enhanced by the constitutive overexpression of mutp53due to their inability to effectively activate MDM2, a negativeregulator of p53 abundance (30, 31). This latter notion could besupported by our finding that cisplatin treatment reduced themRNA level of MDM2 in cells expressing low- and high-risk TP53mutants.

To further assess the effect of suppression of p21 on response tochemotherapy in the EAp53 high-risk mutations, we show in aclonogenic survival assay that HNSCC cells expressing low-riskmutations have an intermediate level of resistance while high-riskTP53mutations have a high level of resistance to platinum-basedtherapy relative to the pBabe vector control, confirming a GOFphenotype with regard to cisplatin sensitivity. The differences incisplatin sensitivity between thewtp53 andmutp53maybedue inpart to the inhibition of accelerated cellular senescence by mutat-ed p53 (24).

Although our initial in vitro experiments suggested a GOFphenotype for high-risk TP53 mutations, our attempt to identifypathways driving this characteristic through expression arrayanalysis revealed that the introduction of high-risk mutationsactually produces a largely nonfunctional p53 (i.e., suppressedlevels of expression of TP53 target genes) that is most similar to acomplete absence of the protein. Furthermore, the low-risk muta-tions have distinct expression variances from cells either lackingp53 or containing a high-risk mutation, which implies thismutation may retain some wildtype functions. The discrepancybetween the expression array data and the in vitro experimentscould be due to different posttranslational modifications of thelow- and high-risk mutations that would not be detected on anmRNAexpression array or an altered protein–protein interactomethrough which p53 gains function through functionally signifi-cant interactions with important cellular protein targets (32, 33).In support of this lattermechanism,wehave recently reported thatGOF p53 mutations can bind to and inactivate, the mastermetabolic regulatory protein, AMPK, which leads to gain ofoncogenic functions (34).

Furthermore, evidence is now accumulating to indicate thatdifferent p53 mutations possess different functions in differenttissues, potentially reflecting differences in the expression oftheir cellular targets (35). Therefore, understanding the con-sequences of each p53 mutation in relationship to diseaseprogression and response to therapy promises to be an extreme-ly complex undertaking and thus highlights the importance ofour current study.

We further characterized not only the functional spectrum ofp53mutations but also the ability of EAp53 to predict response tocisplatin in vivo with an orthotopic nude mouse model of tonguecancer. These results confirmed that tumors bearing high-risk

mutations are more resistant to cisplatin compared with tumorsexpressing either wtp53or low-riskmutant p53. The lack of tumorresponse to treatment in mice with high-risk p53-bearing tumorswas similar to tumors lacking p53 expression, which corroboratesthe expression array data. In addition to having an improvedtumor response, animals with tumors harboring wtp53 and low-risk mutations treated with cisplatin had an improved survivalcomparedwithboth their untreated controls and the animalswithhigh-risk p53 tumors.

Finally, in an effort to evaluate the predictive ability of TP53mutational status stratified by the EA method, we analyzed acohort of patients with locally advanced oral cavity squamouscell carcinoma who had been treated with platinum-basedneoadjuvant chemotherapy followed by surgery (12, 19). Thisanalysis revealed that EAp53 can identify a population ofpatients with high-risk p53 mutations that do not respond toplatinum-based treatment and had decreased overall survival.Although these results are encouraging, there are limitations tothis study. The percentage of patients with wtp53, 62%, ishigher than expected, which could be attributed to either thesensitivity of sequencing TP53 from paraffin tissues or sequenc-ing exons 5–8, as only approximately 80% of mutations occurwithin the DNA-binding domain (36). Therefore, it is possiblethat the portion of the wild-type p53 patients' tumors that didnot have a clinical response actually harbored TP53 mutations.Taken together, these explanations may partly account for notonly the high percentage of wtp53 but also the large number ofwtp53 nonresponders. Nonetheless, EAp53 was predictive oflack of response to platinum-based chemotherapy in patientswith high-risk mutant p53, and identified patients with oralcavity squamous cell carcinoma that have decreased survival.To further validate these clinical findings, additional studies ofp53 mutational status in larger cohorts of HNSCC patients thathave received neoadjuvant platinum-based induction therapyare ongoing.

In summary, the EAp53 model clearly identifies a subset ofhigh-risk TP53 mutations associated with decreased sensitivityto cisplatin in preclinicalmodels. In addition, EAp53 can predictresponse to treatment and more importantly a survival benefitfor a subset of patients treated with platinum-based therapy. Tofully evaluate the role of EAp53 as a predictive biomarker ofplatinum response, prospective clinical trials in which patients'tumors are stratified on the basis of their p53 mutational statusand correlated with the response to treatment and survival arenecessary.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: A.A. Osman, D.M. Neskey, P. Katsonis, A.A. Patel,T.J. Ow, L. Licitra, M. Kimmel, O. Lichtarge, J.N. MyersDevelopment of methodology: A.A. Osman, D.M. Neskey, P. Katsonis, A.A.Patel, M.O. Alves, H.D. Skinner, M. Zhao, M. Kimmel, O. Lichtarge, J.N. MyersAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A.A. Osman, D.M. Neskey, A.A. Patel, A.M. Ward,T.J. Ow, M.O. Alves, C.R. Pickering, M.S. Kies, A. El-Naggar, L. Licitra, P. Bossi,J.N. MyersAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):A.A.Osman,D.M.Neskey, P. Katsonis, T.-K.Hsu, S.C.Hicks, T.O. McDonald, M.O. Alves, C.R. Pickering, E.M. Sturgis, L. Licitra,M. Kimmel, M.J. Frederick, O. Lichtarge, J.N. Myers






Writing, review, and/or revision of themanuscript: A.A.Osman,D.M. Neskey,P. Katsonis, S.C. Hicks, T.J. Ow, H.D. Skinner, E.M. Sturgis, M.S. Kies, L. Licitra,P. Bossi, M. Kimmel, M.J. Frederick, O. Lichtarge, J.N. MyersAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): A.A. Osman, D.M. Neskey, A.A. Patel, A.M.Ward, J.N. MyersStudy supervision: A.A. Osman, A. El-Naggar, M. Kimmel, J.N. MyersOther (performed experiments): A.A. OsmanOther (provided TP53 mutational results found in an Italian series ofHNSCC): F. Perrone

Grant SupportThis work was supported by the University of Texas MD Anderson Cancer

Center PANTHEON program (philanthropic support to J.N. Myers), the NIHSpecialized Program of Research Excellence Grant P50CA097007 (J.N. Myers),the NIH/NIDCR R01 DE14613 and R01 DE024601 (J.N. Myers), Cancer

Prevention and Research Institute of Texas (CPRIT) RP120258 (J.N. Myers),National Research Science Award Institutional Research Training GrantT32CA60374 (J.N. Myers), the NIH Program Project Grant C168485 (J.N.Myers), and the Cancer Center Support Grant CA016672 (J.N. Myers). Thiswork was also supported by NIH R01 GM079656 (O. Lichtarge), R01GM066099 (O. Lichtarge), and NSF CCF 0905536 (O. Lichtarge) and DBI0851393 (O. Lichtarge), and Pharmacoinformatics Training Program of theKeck Center of the Gulf Coast Consortia NIH grant no. 5 R90 DKO71505(P. Katsonis).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received September 19, 2014; revised December 16, 2014; accepted January12, 2015; published OnlineFirst February 17, 2015.

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2015;75:1205-1215. Published OnlineFirst February 17, 2015.Cancer Res Abdullah A. Osman, David M. Neskey, Panagiotis Katsonis, et al. Platinum Response in Head and Neck Cancer Patients

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