Home | Clinical Cancer Research - Analytic, Preanalytic, and … · Prostate Cancer Liana B. Guedes1, ... as any p53 nuclear labeling in >10% of tumor cells. Fifty-four formalin-ﬁxedparafﬁnembedded(FFPE)celllinesfromtheNCI-60

Personalized Medicine and Imaging

Analytic, Preanalytic, and Clinical Validation ofp53 IHC forDetectionofTP53MissenseMutation inProstate CancerLiana B. Guedes1, Fawaz Almutairi1, Michael C. Haffner1, Gaurav Rajoria2,Zach Liu2, Szczepan Klimek2, Roberto Zoino2, Kasra Yousefi3, Rajni Sharma1,Angelo M. De Marzo1,4,5, George J. Netto1,William B. Isaacs4,5, Ashley E. Ross4,Edward M. Schaeffer4, and Tamara L. Lotan1,5

Abstract

Purpose: TP53 missense mutations may help to identify pros-tate cancer with lethal potential. Here, we preanalytically, ana-lytically, and clinically validated a robust IHC assay to detectsubclonal and focal TP53 missense mutations in prostate cancer.

Experimental Design: The p53 IHC assay was performed in aCLIA-accredited laboratory on the Ventana Benchmark immu-nostaining system. p53protein nuclear accumulationwas definedas any p53 nuclear labeling in >10% of tumor cells. Fifty-fourformalin-fixed paraffin embedded (FFPE) cell lines from the NCI-60 panel and 103 FFPE prostate cancer tissues (88 primaryadenocarcinomas, 15 metastases) with known TP53 mutationstatus were studied. DU145 and VCaP xenografts were subjectedto varying fixation conditions to investigate the effects of pre-analytic variables. Clinical validation was performed in twopartially overlapping radical prostatectomy cohorts.

Results: p53 nuclear accumulation by IHC was 100% sensitivefor detection of TP53 missense mutations in the NCI-60 panel(25/25 missense mutations correctly identified). Lack of p53nuclear accumulation was 86% (25/29) specific for absence ofTP53 missense mutation. In FFPE prostate tumors, the positivepredictive value of p53 nuclear accumulation for underlyingmissense mutation was 84% (38/45), whereas the negative pre-dictive value was 97% (56/58). In a cohort of men who experi-enced biochemical recurrence after RP, the multivariable HR formetastasis among caseswith p53nuclear accumulation comparedwith those withoutwas 2.55 (95% confidence interval, 1.1–5.91).

Conclusions: IHC is widely available method to assess for thepresence of deleterious and heterogeneous TP53 missense muta-tions in clinical prostate cancer specimens. Clin Cancer Res; 23(16);4693–703. �2017 AACR.

IntroductionBoth older and more recent sequencing studies of castrate-

resistant prostate cancer (CRPC) metastases have revealed ahigh frequency of TP53 mutations in advanced disease, makingthese genomic alterations among the most enriched whencomparing primary and advanced metastatic prostate cancer(1–4). Although TP53 aberrations are relatively rare in primarytumors, occurring in less than 10% (5), there is a 4- to 5-foldincrease in alterations in CRPC (4) and TP53 and RB1 muta-

tions are considered key drivers of small-cell neuroendocrinecarcinomas in the prostate and other organs (6, 7). The reasonfor this striking and unanticipated enrichment of TP53 loss inaggressive prostate cancer is currently being scrutinized by anumber of groups. In two recent studies, combined loss of TP53and RB1 facilitated prostate cell lineage plasticity and anti-androgen resistance in vitro and in vivo, in part due to down-stream SOX2 activation (8, 9); however, these mechanisticstudies focused on genomic deletion of TP53, rather thanmutation. Interestingly, in both primary and metastatic pros-tate tumors, TP53 alterations are relatively evenly split betweenthose resulting in pure loss-of-function for the tumor suppres-sor protein (nonsense, frameshift, splice site mutations, andhomozygous deletion) and missense mutations which result inpotential gain-of-function, loss-of-function, and/or dominantnegative phenotypes (4, 5). The differing cellular effects of thesedisparate types of alterations in TP53 remains a hotly debatedtopic in cancer (10) and whether each has different associationswith prostate cancer clinical outcomes remains unknown. Fur-ther, although TP53 is thought to be a relatively early occurringmutation in primary prostate cancer (11–14), the chronicity ofthis alteration with respect to other common genomic changesin prostate cancer, and its potential heterogeneity in primarytumors has not been elucidated.

Underlying these knowledge gaps in prostate cancer is the factthat few validated assays exist to query TP53 status in prostatecancer. Although sequencing will clearly remain a gold-standard

1Department of Pathology, Johns Hopkins University School of Medicine,Baltimore, Maryland. 2Pathline Emerge Pathology Services, Ramsey, New Jer-sey. 3GenomeDx Biosciences, Vancouver, British Columbia. 4Department ofUrology, Johns Hopkins University School of Medicine, Baltimore, Maryland.5Department of Oncology, Johns Hopkins University School of Medicine, Balti-more, Maryland.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Current address for G.J. Netto: Department of Pathology, University of AlabamaMedicine, Birmingham, Alabama.

Corresponding Author: Tamara L. Lotan, Johns Hopkins University School ofMedicine, CRB II Rm 343, 1550 Orleans Street, Baltimore, MD 21231. Phone: 410-614-9196; Fax: 410-614-0671; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-17-0257

�2017 American Association for Cancer Research.

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technique, recent work has shown that TP53 mutations can beheterogeneous and extremely focal in primary prostate tumors,requiring ultra-deep sequencing to detect (12). Thus, a less expen-sive in situ assay to rapidly assess TP53 status in large portions oftissue would be useful, both for routine clinical screening ofprimary tumors where alterations may be focal, and to begin toaddress some of the biological questions outlined above. Impor-tantly, missense mutations in TP53 generally occur in a fewhotspots in the DNA-binding domain of the protein, and havelong been recognized to result in stabilization and resultantnuclear accumulation of the p53 protein in tumor cells (15–18). Although a complete mechanistic understanding has beenelusive (19, 20), this phenomenon is useful because p53 nuclearaccumulation by IHC can be a surrogate to predict TP53 genestatus in situ (21). p53 IHC assays have been used previously inother tumor types (22, 23) and prostate cancer (2, 24–31) forsome time to screen for TP53 missense mutations; however,genetically validated assays for use in prostate cancer have notbeen developed using contemporary, automated, and clinical-grade IHC platforms.

Here, we extensively analytically, preanalytically, and clinicallyvalidated a clinical-grade IHC assay to screen for presence ofstabilizing TP53 missense mutations in prostate cancer in aCLIA-accredited laboratory environment. We show that this assayis highly sensitive and specific for detection of TP53 missensemutations in cell lines and primary prostate tumors, the assay isrobust to preanalytic fixation conditions and could be of partic-ular use to identify heterogeneous, subclonal TP53 aberrations inthe setting of prostate biopsies that may be difficult to detectwithout ultradeep sequencing. Finally, we show that TP53 status,as determined by this assay, is associated with risk ofmetastasis intwo large cohorts of surgically treated prostate cancer patients.

Materials and MethodsPatients and tissue samples

In accordance with the U.S. Common Rule and after IRBapproval with a waiver of consent, a total of 103 prostate tumorswith known TP53 sequencing results (including 90 primarytumors at radical prostatectomy, transurethral resection of pros-tate or needle biopsy and 13 autopsy specimens) from several

different cohorts were utilized for the validation study. The firstgroup consisted of 28 cases from a radical prostatectomy case–cohort design (described below as used for outcomes analysis;refs. 32, 33). These samples were immunostained for p53 ontissue microarrays (TMA; see below) and 28 cases (including fournegative for p53 nuclear accumulation and 24 positive for nuclearaccumulation) were selected for sequencing as described belowafter examination of immunostaining for p53. The second cohortincludedmetastatic tissue from13 autopsy cases sampled onTMAfor immunostaining, for which previously published TP53sequencing data were available (11, 34). The third cohort includ-ed four small cell carcinomaof the prostate cases sampled onTMAfor which previously collected TP53 sequencing data were avail-able (6). The fourth cohort included two radical prostatectomycases (standard slides) with known homozygous deletions ofTP53 from prior studies (35). Finally, the last cohort included56 radical prostatectomy cases from patients who subsequentlydeveloped castration-resistant metastatic disease and receivedenzalutamide and/or abiraterone at Johns Hopkins between2010 and 2015. These 56 patients included all patients with aradical prostatectomy at Johns Hopkins and available tissue forsequencing, selected from a retrospective analysis of 309 conse-cutive patients treated with first-line abiraterone or enzalutamideat Johns Hopkins during this time period. (Maughan and collea-gues; unpublished data). For these cases, all tumors weresequenced irrespective of p53 immunostaining status. The detailsof each cohort are included in Supplementary Table S1.

To assess the frequency of p53 nuclear accumulation by IHCin prostate needle biopsies, we examined p53 IHC in a cohortof 40 Gleason score 9 or higher needle biopsies describedpreviously (36).

For outcomes analysis, we selected a total of 456 primaryprostate tumors from two overlapping and previously publishedradical prostatectomy cohorts at Johns Hopkins (including 28tumors with sequencing data as described above; refs. 32, 33).One cohort included 322 patients using a nested case–cohortdesign for metastasis, where the time to clinical metastasis wasmeasured from the time of diagnosis. An additional and highlyoverlapping cohort (�55% overlap) contained 195 men who allexperienced biochemical recurrence (defined as a PSA measure-ment of �0.2 ng/mL on two separate occasions) after radicalprostatectomy. Here, time to metastasis was measured from timeof biochemical recurrence. These cohorts were originally designedto test for prognostic tumor biomarkers and were accordinglyhighly enriched for adverse oncologic outcomes. Tumor tissuefrom thedominant tumor nodule andbenign tissuewere sampledin quadruplicate on 16 individual TMAs utilizing 0.6 mm cores.

Cell line TMAA total of 56 cell lines from the NCI-60 cell line panel (Devel-

opmental Therapeutics Program, NCI) were used to evaluate p53IHC staining. All cell lines were pelleted, fixed in 10% neutral-buffered formalin, and processed and cut as tissue. Cell lines werepunched and TMAs created as previously described (37). Addi-tional prostate cell lines with known TP53 gene status were alsostudied, including RWPE-1, VCaP, DU145, and PC-3 (ATCC).RWPE-1 cells havewild-typeTP53. VCaP andPC3 cells harbor onedeleted allele of TP53 and one missense mutation or frameshiftmutation, respectively (R428W and A138fs; refs. 38, 39). DU145cells harbor missense mutations on both alleles (V274Fand P223L; ref. 38). For all prostate cancer cell lines, cell line

Translational Relevance

TP53mutations are among the most highly enriched geno-mic alterations among castrate-resistant prostate cancers(CRPC) compared with primary tumors and recent preclinicaldata suggests that TP53 inactivation may cooperate with otheralterations to confer lineage plasticity and androgen indepen-dence in the prostate. We validated an in situ assay to sensi-tively and specifically detect TP53 missense mutations inprostate cancer using IHC. This assay, which is robust tofixation conditions, is particularly useful to screen for focalor subclonal mutations that could be missed by sequencingandwhichwere present in nearly half of all primary tumors. Intwo overlapping radical prostatectomy cohorts, presence ofTP53 missense mutation by IHC was associated with devel-opment of metastasis, clinically validating the assay for use inhigh risk prostate cancer or future clinical trials.

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authenticity and mycoplasma contamination were routinelyassessed in 6- to 10-month intervals by short tandem repeat (STR)genotyping and PCR-based testing, respectively (last testedDecember 2015). For the remaining NCI-60 cell lines, STR geno-typing was completed once prior to creation of the cell line TMA.

Xenograft TMAVCaP and Du145 cell line xenografts were established as

previously described (40) and subjected to different fixationconditions after excision to evaluate the effect of preanalyticvariables on the performance of the p53 IHC stain. After harvest-ing, the xenograft tumors were sectioned into three fragments andthen divided in five groups, each subjected to varying intervals ofcold ischemia at room temperature (0, 2, 4, 12 or 24 hours) priorto fixation. After the appropriate period of cold ischemia elapsed,the thirds of the tumor specimen with up to 4 hours of ischemiawere put into 10% neutral-buffered formalin and fixed for 4, 12,or 24 hours. The thirds of the specimen with periods of ischemialonger than 12 hours were left in fixation for 12, 24, 36, or 48hours intervals. The processed samples were arranged in a TMAand stained for p53 as below.

Detection of p53 nuclear accumulation by IHCp53 IHC was performed on the Ventana Benchmark autostain-

ing system using a mouse monoclonal antibody (BP53-11) afterantigen retrieval in CC1 buffer followed by detection with theiView HRP system (Roche/Ventana Medical Systems). This pro-tocol was previously validated for detection of p53 mutations inovarian carcinoma (22) and the BP53-11 clone was tested inprostate and showed very low background staining in benignglands in comparison with another available clone from Ventana(D07), facilitating detection of nuclear p53 accumulation. EachTMA spot containing tumor cells was visually dichotomouslyscored for presence or absence of nuclear p53 signal by a urologicpathologist blinded to the gene expression data (TLL). A spot wasconsidered to show p53 nuclear accumulation if >10% of tumornuclei showed p53 positivity. This cutoff value was chosenbecause it was the most correlated with presence of wild-typeTP53 in a prior genomic validation of this staining protocol inovarian carcinoma (22) and themost commonly chosen cutoff inameta-analysis of p53 staining inother tumor types (23). A tumorwas considered to showp53 nuclear accumulation if any sampledspot was scored as p53 positive, and as p53 negative if all sampledspots were scored as p53 negative.

TP53 sequencingFor samples from the TMAs, a total of five 0.6 mmpunches were

procured from the same area in the paraffin block sampled on theTMA. DNA was extracted from these FFPE cores using the QiagenAllPrep DNA Extraction Kit (Hilden) according to the manufac-turer's directions. DNA concentrations were quantified with theQubit fluorometer, using a Quant-iT dsDNA High SensitivityAssay Kit (Invitrogen). Mutations were screened by next-genera-tion sequencing (NGS) using multiplexed PCR (AmpliSeq Hot-spot Panel) to generate libraries. Adapters were ligated to the PCRproducts, where the sequences are taggedwith a specific barcodes.The barcoded libraries were clonally amplified using emulsionPCR (emPCR). The emPCR product was purified using magneticbead purification followed by semiconductor-based sequencingon an Ion Torrent PGM (Thermo Fisher). For each gene, theminimum required coverage was 500 sequence reads based on

bidirectional sequencing. The minimum acceptable allelic fre-quency was 5%, as any lower values may be background noise.Each variant was analyzed manually using variant caller from IonTorrent software and cross-referenced with Ingenuity software forbioinformatics (Qiagen). The amplicons included for sequencingin the Ampliseq Hotspot panel included 91% (68/75) of themissense mutations detected in the AACR Project GENIE data forprostate cancer (41), although seven of these detectable muta-tions (9%) were only detectable at the end of the amplicon sowould require sequencing validation for reporting by a CLIAlaboratory.

Disruptive versus nondisruptive TP53mutation categorizationAs defined by Poeta and colleagues (42, 43), disruptive muta-

tions include (i) any mutation that originates a stop codon(nonsense, frameshift, and intronic); (ii) missense mutationslocated inside the L2 or L3 loops (codons 163–195 or 236–251) replacing one residue by another of a different polarity orcharge; and (iii) in-frame deletions within the L2 or L3 loops.Nondisruptive mutations are all those not classified as disruptiveand include (i) missense mutations and in-frame deletions locat-ed outside the L2–L3 loops and (ii) missense mutations withinthe L2–L3 loops but replacing one residue with another of thesame polarity or charge. Disruptive TP53 mutations lead tocomplete or almost complete predicted loss of activity of thep53 protein, in contrast to nondisruptive mutations that mayretain some p53 function or have gain-of-function properties. ThePoeta and colleagues criteriawereused to categorize all discoveredTP53mutations in FFPE samples by disruptive versus nondisrup-tive status and this classification is indicated in SupplementaryTable S3.

Statistical analysisThe significance level was 0.05 for all statistical tests and

analyses were performed in R v3.1 (R Foundation). Primaryendpoint of the study was defined as metastases evidenced byaxial imaging (CT or MRI) or nuclear medicine bone scan. Todetermine the association of clinicopathologic variables with p53status, Fisher exact test was used. For the intermediate/high riskcohort, time to event was defined as time from radical prostatec-tomy to last follow-up or metastasis. For the biochemical recur-rence (BCR) cohort, time to eventwas defined as time fromBCR tolast follow-up or metastasis. Because of the case–cohort nature ofour study, univariable and multivariable Cox proportionalhazards model using the Lin-Ying method was used to evaluatethe performance of p53 status in predicting metastasis (44, 45).Cumulative incidence curves were constructed using Fine-Graycompeting risks (where deaths from other causes was consideredas a competing risk) analysis with appropriate weighting of thecontrols to account for the case–cohort study design (44, 45).

Resultsp53 nuclear accumulation by IHC correlates withpresence of underlying TP53 missense mutation in NCI-60cancer cell line panel

To begin to determine the sensitivity and specificity of the p53IHC assay, we first examined a previously described TMA com-posed of 54 of the 56 cell lines from the NCI-60 cell line panel(IGROV-1 and CAKI-1 were missing). In the cell line TMA, p53protein nuclear accumulation was 100% sensitive for detection ofdeleterious TP53missensemutations, detecting 25 of 25missense

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mutations correctly (Fig. 1A; Tables 1A and 2 and SupplementaryTable S2). The specificity of the IHC assay was 86%, with lack ofp53 nuclear accumulation in 25 of 29 cell lines that lacked

missense mutations (i.e., wild-type TP53 or loss-of-functionmutations in TP53). Of the four discordant cell lines, two hadpossible frameshift mutations in TP53 (COLO-205 and SF-539)with additional missense mutations also reported in some stud-ies. Both of these cell lines were classified as having inconclusiveTP53 status (http://p53.free.fr/Database/Cancer_cell_lines/p53_cell_lines.html). The other two discordant lines with p53nuclear accumulation had reported wild-type TP53 status (UO-31) and a reported splice-site mutation (OVCAR-8, p.Y126_splice; ref. 46). The positive predictive power (PPV) of

Figure 1.

Representative p53 immunostaining in FFPE prostate cell lines and primary and CRPC prostate tumors. A, RWPE-1 cells, which are TP53wild-type (WT), show rarepositive nuclei comprising less than 10% of the tumor on p53 immunostaining. In contrast, DU145 cells (with two mutations, p.V274 and p.P223L) and VCaPcells (p.R248W)withmissensemutations show robust nuclear accumulation of p53. PC3 cells, which have a loss-of-function frameshift mutation (p.A138fs) and LOHare entirely negative for p53 expression. All photomicrographs are reduced from 200�. B, Only rare, weakly positive nuclear immunostaining for p53 is seenin benign prostate tissues and primary and CRPC tumors that are TP53 WT, although some cytoplasmic staining is seen specifically in these tumors of unknownsignificance. The lack of nuclear positivity in benign prostate tissue and TP53 WT tumors meant that the IHC assay cannot distinguish tumors that are TP53WT from those with loss-of-function alterations (homozygous deletion, frameshift, splice site, or nonsense mutations). In contrast, tumors with missense mutationsof TP53 (p.R175H, p.R273C, p.V157A, p.131N) are readily distinguishable from WT and show robust nuclear accumulation of p53 protein in the majority ofcells. All photomicrographs are reduced from 200�.

Table 1A. Correlation between p53 nuclear accumulation by IHC and presenceof TP53 missense mutation in NCI-60 cell line panel

p53 nuclearaccumulation

Normalp53 IHC

TP53 missense mutation 25 0Absence of TP53 missense mutation 4 25

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http://p53.free.fr/Database/Cancer_cell_lines/p53_cell_lines.html

http://p53.free.fr/Database/Cancer_cell_lines/p53_cell_lines.html


p53 IHC for detecting TP53missense mutation was 86% (25/29)and the negative predictive power (NPV) of lack of p53 nuclearaccumulation for lack of underlying TP53 mutation was 100%(25/25).

Of note, in cell lines, complete lack of p53 expression by IHCgenerally did distinguish tumors with loss-of-function mutationsand/or homozygous deletion in TP53 (n ¼ 8) from those withwild-type TP53, which showed nuclear staining for p53 in fewerthan10%of cells (n¼13; Fig. 1A, compare RWPE-1 andPC3 cells;Supplementary Table S2). However, there were a few exceptionsto this observation. Two cell lines (CAKI-1 andOVCAR-5) showedcomplete absence of p53 by IHC despite a reported wild-typeTP53 locus (46). In addition, two other cell lines (MOLT-4 andSN12C) showed some weak nuclear positivity for p53 despite thepresence of reported underlying nonsensemutations (p.306� andp.E336�, respectively; ref. 46).

p53 nuclear accumulation by IHC is highly associated withpresence of underlying TP53 missense mutation in formalin-fixed paraffin embedded prostate tumors

The sensitivity and specificity of the p53 IHC assay were thenevaluated in 103 formalin-fixed paraffin embedded (FFPE) pros-tate tumors (88 primaries and 15 metastases) with paired DNAsequencing and IHC results in FFPEmaterial. Importantly, for 101of 108 tumors, p53 immunostaining was initially evaluated onTMA spots, whereas the remainder were evaluated on standardhistologic sections. The sensitivity of p53 IHC nuclear accumu-lation was 95% for missense mutation detection, with IHCnuclear accumulation detecting 38 out of 40 missense mutations(Fig. 1B; Tables 1B and 2; Supplementary Table S3). Using IHC,one of two missed mutations was p.R282W in a radical prosta-tectomy specimen where tissue preservation appeared to be poor(Supplementary Fig. S1). Of interest, that same mutation waspresent and successfully detected by IHC in a separate, unrelatedsample, where the p53 immunostaining showed heterogeneousp53 overexpression (Supplementary Fig. S2). The other discor-dant case was an unusual case with eight individual missensemutations in a single radical prostatectomy specimen, includingp.R267W, G226D,M160I, E349K, E339K, A129T, E11K and V10I(Supplementary Fig. S1; Supplementary Table S2). This tumorwas Gleason Score 3 þ 5 ¼ 8 (using ISUP 2005 criteria) andpT3a and occurring in a 59-year-old white male. The tumoritself did not look morphologically unusual, although did havea very high-grade tumor component. It is tempting to speculatethat it could be a hypermutated tumor, however it was MSH2intact by IHC as well. We also cannot rule out the possibility of a

sequencing artifact in this case. Indeed, only one other samplehad more than one TP53 mutation detected (with 4 total, also aradical prostatectomy).Mostmutations detected by the IHC assaywere in the DNA-binding domain of p53 as expected (Supple-mentary Fig. S3).

Taken together, the specificity of the IHC assay was 89%, with56/63 tumors lacking TP53 missense mutations by sequencingalso lacking p53 nuclear accumulation by IHC. Of the sevendiscordant cases lacking TP53 missense mutations, but showingnuclear accumulation of p53 on IHC, five had wild-type TP53 bysequencing and two had in-frame deletions in the DNA bindingdomain (p.I255delI, and p.V203_D207del15; SupplementaryFig. S1 and Supplementary Table S3). Of the five cases thatshowed p53 nuclear accumulation with wild-type TP53, threetumors had p53 immunostaining reexamined on standard sec-tions to evaluate whether tumor heterogeneity could account fordiscordance. Of note, in all of these cases, heterogeneity of p53expression could be observed, raising the possibility of focal TP53mutations in a tumor subclone that may not have been detectedby the sequencing assays (Fig. 2). Although we cannot entirelyexclude the possibility that the Ampliseq sequencing assay failedto detect underlying missense mutations in these cases due to itshotspot design, it is notable that this assay was designed tothoroughly cover the TP53 gene, capable of detecting 91% of themissense mutations detected in the AACR Project GENIE prostatecancer sequencing database (41). Overall, the negative predictivevalue (NPV) of lack of IHC nuclear accumulation for lack of TP53missense mutation was 97% (56/58) and the positive predictivevalue of p53 nuclear accumulation for presence of underlyingTP53mutation was 84% (38/45). In contrast to our observationsin cell lines above, complete lack of p53 expressionby IHCdid notdistinguish tumors with loss-of-function mutations and/orhomozygous deletion in TP53 (n ¼ 12) from those with wild-type TP53 (n¼ 44) because the basal level of nuclear p53 proteinexpression in most wild-type prostate tumors was extremely lowto undetectable (Fig. 1; Supplementary Fig. S1).

Previous work has categorized TP53 mutations as "disruptive"versus "nondisruptive" (42, 43), Disruptive TP53mutations leadto complete or almost complete predicted loss of activity of thep53 protein, in contrast to nondisruptive mutations that mayretain some p53 function or have gain-of-function properties.Interestingly, the majority of the nondetected alterations by p53IHC accumulation were disruptive (80% or 8/10; SupplementaryTable S3), whereas the majority of the detected mutations werenondisruptive by these criteria (76% or 19/25), suggesting thatour assay is biased towards mutations that may have gain-of-function properties (as has been previously described formany ofthese missense mutations).

Frequency of p53 nuclear accumulation amongGleason score 9prostate biopsies

Finally, because the biopsy setting is likely the most clinicallyuseful context to apply the p53 IHC assay, we assessed thefrequency of p53 nuclear accumulation among a cohort of

Table 2. Performance characteristics of p53 IHC compared to TP53 sequencing results

Sensitivity Specificity PPV NPV

NCI-60 cell lines 100% (25/25) 86% (25/29) 86% (25/29) 100% (25/25)FFPE prostate tumors 95% (38/40) 89% (56/63) 84% (38/45) 97% (56/58)

Table 1B. Correlation between p53 nuclear accumulation by IHC and presenceof TP53missensemutation in formalin fixed paraffin embedded prostate tumors

p53 nuclearaccumulation

Normalp53 IHC

TP53 missense mutation 38 2Absence of TP53 missense mutation 7 56






40high-risk biopsieswithGleason score 9 or higher tumor.Of the40 tumors selected for IHC, 39 (97.5%) had sufficient tumor foranalysis. Of these, 13% (5/39) had p53 nuclear accumulationpresent. Interestingly, of the positive cases, 80% (4/5) had focalnuclear accumulation at biopsy (Fig. 2C and D).

p53 nuclear accumulation by IHC is minimally affected bydifferent preanalytic variables of delay of fixation or time infixation

VCaP and Du145 cell line xenografts, which both have mis-sensemutation of TP53 (38, 39) andnuclear accumulation of p53protein, were used to validate the robustness of the p53 IHC assayto preanalytic variables, including time of fixation and coldischemia time. Time in formalin fixation did not markedly affectintensity of nuclear accumulation of p53 on IHC in DU145xenografts, although minimal decrease in intensity was noted forVCaP xenografts. Similarly, cold ischemia time did not appreci-ably affect intensity of the p53 IHC signal or number of cellsstaining in either xenograft, although increased fixation delay didresult in apparent tissue degeneration and autolysis and increasedbackground signaling in both lines (Fig. 3).

p53 nuclear accumulation by IHC correlates with risk ofmetastasis in two cohorts of surgically treated patients

Having fully analytically validated the p53 IHC assay, wenext sought to clinically validate it. We assessed whether p53nuclear accumulation by IHC is associated with metastaticprogression in two partially overlapping and previouslyreported radical prostatectomy cohorts with clinical follow-up(32, 33). The first cohort included 322 patients in a nestedcase–cohort design for metastasis. Demographic and clinical–pathologic characteristics of the cohort are reported in Supple-mentary Table S4. This cohort included 27 tumors with p53nuclear accumulation (8%). Because the immunostains wereperformed on TMAs, we first examined whether tumor sam-

pling in TMA format might miss p53 nuclear accumulation incomparison to immunostaining on standard slides. We immu-nostained 49 cases from the cohort that lacked p53 nuclearaccumulation on TMA spots, examining the same dominantnodule that was punched for the TMA using standard sections.None of the cases showed focal p53 nuclear accumulation instandard sections, suggesting that TMA sampling is reasonablysensitive to detect p53 nuclear accumulation and suitable forusing in outcome studies. In the TMA cohort, p53 nuclearaccumulation was highly enriched among high Gleason scoretumors compared with lower Gleason score tumors, withnuclear accumulation in 20% of Gleason score 9–10 tumors(P < 0.001). In univariable analyses, Gleason score, positivesurgical margin status, tumor stage, and p53 IHC status (Fig.4A) were all associated with risk of metastasis, whereas patientage and preoperative PSA were not significantly associated withoutcome (Table 3). The univariable HR for metastasis amongcases with p53 nuclear accumulation compared with thosewithout was 4.84 [95% confidence interval (CI), 2.44–9.61].In multivariable models, only Gleason score, surgical marginstatus, seminal vesicle invasion, and lymph node involvementremained significantly associated with risk of metastasis. TheHR for metastasis among men with p53 nuclear accumulationcompared with those without was 1.87; however, this trend didnot reach statistical significance (95% CI, 0.83–4.23; P ¼0.13; Table 3).

We next examined the association of p53 IHC status withmetastasis in a partially overlapping cohort of 195 men whounderwent radical prostatectomy followed by biochemical recur-rence (Supplementary Table S5). In this cohort, p53 nuclearaccumulation was present in 9% (17/195) of cases, andwas againenriched among Gleason score 9–10 tumors (seen in 20%)compared with lower Gleason score tumors. In this cohort,univariable analyses showed that Gleason score, seminal vesicleinvasion, lymph node involvement, and p53 IHC status (Fig. 4B)

Figure 2.

Heterogeneous p53 immunostainingon standard histologic sections wascommon in primary tumors withdiscordant TP53 sequencing and p53immunostaining results. A, A tumorthat was TP53 WT by sequencingshows focal p53 nuclear accumulationon standard section (arrow, top left)with an adjacent area lacking nuclearaccumulation (arrow, bottom right).B,A tumor that was TP53 WT bysequencing because a p.C135Ymutation was detected in <5% of cells,shows focal p53 nuclear accumulationon standard section (arrow, top right)with an adjacent area lacking nuclearaccumulation (arrow, lower left). Cand D, Gleason score 9 biopsies withclear focal nuclear accumulation ofp53 in some, but not all tumor cells(arrows). All photomicrographs arereduced from 200�.

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were associated with risk of metastasis, while patient age,preoperative PSA, extraprostatic extension, and surgical mar-gins were not significantly associated with outcome (Table 4).The univariable HR for metastasis among cases with p53nuclear accumulation compared with those without was 4.14(95% CI, 2.41–7.11). In multivariable analyses, only Gleasonscore, seminal vesicle invasion, and p53 nuclear accumulationremained significantly associated with risk of metastasis. Themultivariable HR for metastasis among cases with p53 nuclear

accumulation compared with those without was 2.55 (95% CI,1.1–5.91; P ¼ 0.03; Table 4).

DiscussionHerein, we have extensively preanalytically, analytically, and

clinically validated a clinical-grade and automated IHC assay foridentifying the presence of deleterious TP53 missense mutationsinprimary prostate tumors. Inoneof the largest studies inprostate

Figure 3.

Effect of preanalytic tissue fixationconditions (cold ischemia time andtime in fixation) on p53immunostaining in two xenograftmodels with known TP53 missensemutations. A, Effect of variation offixation duration (4, 12, and 24 hours in10% neutral-buffered formalin withimmediate fixation) on p53immunostaining in DU145 and VCaPxenografts. Minimal variation in p53nuclear accumulation is seen in DU145cells (left panels), whereas VCaP cellsshow a slight decrease in intensity ofstainingwith longer fixation conditions(right), although number of cellsstaining is not markedly affected. B,Effect of variation of cold ischemiaduration (4, 12, 24 hours withoutfixation at room temperature with 24-hour fixation in 10% neutral-bufferedformalin) on p53 immunostaining inDU145 and VCaP xenografts. Minimalvariation in intensity of p53 nuclearaccumulation is seen in DU145 cells(left) or VCaP cells (right) withvariation of cold ischemia time,although tissue quality clearlydecreaseswith increased autolysis andbackground staining compared with 0hours old ischemia time (comparewithlast row of both panels in A).






cancer to compare an IHCassaywithNGSdata, we found that p53IHC is 95% sensitive and nearly 90% specific for TP53 missensemutation detection compared to TP53 sequencing in over 100prostate tumors. Importantly, the IHC assay is fairly robust topreanalytic variables such as cold ischemia time and tissue fixa-tion duration. For clinical validation of the assay, we show thatnuclear accumulation of p53 protein is robustly associated withpoor outcomes in two overlapping clinical cohorts of surgicallytreated primary prostate tumors. Given the high sensitivity andspecificity of the IHCassaywith respect to sequencing,we proposethat p53 IHC is a cost-effective and widely available method toscreen primary prostate tumors for prediction of aggressivenessand outcome and is feasible to perform in high risk prostatebiopsies. In addition, IHC provides an invaluable in situ technol-ogy to screen for focalTP53mutations. Recent ultra-deep sequenc-ing and clonal evolution studies have suggested that TP53 muta-tions, although generally occurring early in the primary tumor(11–14), may be quite focal and heterogeneous in primaryprostate tumors (12, 13, 47). Indeed, in this study, we have alsoobserved frequent heterogeneity of p53 nuclear accumulation in

as many as 40% of tumors. Although tumor heterogeneity is achallenge for any genomic technology, IHC is perfectly suited toscreen large areas of tumor for focal alterations at a low cost, andthuswouldbequite useful in this regard inheterogeneous tumors.

The TP53 gene is altered in nearly 50% of human tumorsoverall, giving it the dubious distinction of being the mostfrequently mutated gene in human cancer (48). However, com-pared with other epithelial tumors, TP53 mutation is actuallyrelatively infrequent in prostate cancer, with the prevalence hov-ering around 7% of contemporary surgical cohorts such as theTCGA cohort, with an additional 1% or so of tumors harboringhomozygous deletions involving TP53 (5). The low rate of TP53alteration in prostate cancer may be a reflection of the generallylower mutational burden seen in this tumor type compared withother carcinomas. Interestingly, however, in recent sequencingefforts in CRPC, the rate of TP53 mutations was dramaticallyincreased compared with what was observed in the primarysetting, with up to 40%of CRPC tumors showing TP53mutationsand another 10% or so showing homozygous deletions or geno-mic rearrangements involving TP53 (4). This significant

Figure 4.

Cumulative incidence of metastasis in patients from intermediate and high risk prostate cancer case-cohort study (A) or post-biochemical recurrence case–cohortstudy (B), stratified by p53 immunostaining status. Note that the case–cohort design makes a random sampling of the cohort and then is enriched withall the metastatic patients (not selected randomly). Thus, to get the full cohort, the controls are reweighted by the inverse of sampling fraction (32, 33).

Table 3. Univariable (UVA) andmultivariable (MVA)models of association of p53 and clinicopathologic risk factors withmetastasis in intermediate/high risk radicalprostatectomy cohort

UVA MVAVariables HR (95% CI) P HR (95% CI) P

Patient age at RP 0.99 (0.96–1.02) 0.52 1.00 (0.96–1.04) 0.93Log2 preoperative PSA (ng/mL) 1.23 (0.94–1.61) 0.13 1.10 (0.84–1.42) 0.49RP Gleason score �3þ4 Ref 1 ref 1RP Gleason score 4þ3 1.90 (0.89–4.04) 0.1 1.81 (0.83–3.95) 0.14RP Gleason score 8 4.70 (2.24–9.89) <0.001 3.09 (1.26–7.56) 0.01RP Gleason score �9 13.00 (7.19–23.51) <0.001 7.12 (3.82–13.29) <0.001Positive surgical margins 2.08 (1.34–3.24) 0.001 1.79 (1.07–2.99) 0.03Extraprostatic extension 3.95 (2.17–7.19) <0.001 1.43 (0.72–2.86) 0.31Seminal vesicle invasion 7.96 (5.04–12.58) <0.001 3.32 (1.91–5.77) <0.001Lymph node invasion 6.66 (4.12–10.75) <0.001 2.63 (1.43–4.83) 0.002p53 normal Ref 1 Ref 1p53 nuclear accumulation 4.84 (2.44 -9.61) <0.001 1.87 (0.83–4.23) 0.13

Guedes et al.





enrichment of TP53 alterations in CRPC compared with primarytumors was rivaled only by that seen with alterations in theandrogen receptor (AR), where genomic alterations are directlyand heavily selected for after the imposition of hormonal ther-apies. Given that TP53 mutations generally occur early in tumorprogression, occurring in the primary tumor before clonal diver-gence in the metastatic setting, these data suggest that primarytumors with TP53 alterations very likely have an unusuallyaggressive clinical course, which could potentially be predictedfrom an early timepoint in clinical care.

Of interest, in both the primary and metastatic settings, TP53mutations are fairly evenly split in prostate cancer betweenmissense mutations and nonsense/frameshift/indel mutations(4, 5). This is in stark contrast to other carcinomas, wheremissense mutations are more heavily favored, comprising overthree quarters of catalogued mutations, compared with non-sense/frameshift/indel alterations (49). Although it has beendebated for some time, it is increasingly clear in other tumortypes that TP53missense mutations may be a fairly unique classof alteration associated with gain-of-function, rather than sim-ple loss-of-function or dominant-negative phenotypes as wasoriginally thought (10, 50–53). Indeed, even the subtype ofmissense mutation may confer specific transcriptional or othergain-of-function activities (10, 54). However, this remains to beproven for prostate cancer, and it is currently unknown whetherthe clinical outcomes of patients with TP53 nonsense/frame-shift/indel alterations differ significantly from those with mis-sense mutations. p53 missense mutation, as detected by theIHC assay described in this study, is clearly associated withadverse outcomes in surgical cohorts of prostate cancerpatients. Whether TP53 loss-of-function alterations (such asnonsense/frameshift/indel mutations that are not detected bythe current IHC assay) have a similar prognosis is unclear.Future studies will examine these questions in cohorts withextensive clinical follow-up.

One clear limitation of the IHC assay described herein is itsinability to detect nonsense/frameshift/indel alterations in TP53,likely due to the relatively lowoverall expression of p53 protein inbenign prostate glands and those tumors lacking missense muta-tions.Of interest, the same assay did successfully detectmutationsresulting in loss of protein in ovarian carcinomas, perhapsbecause the endogenous expression of wild-type p53 protein inthe ovary is relatively higher than that seen in the prostate,enabling more sensitive detection of protein loss (22). Along thesame lines, we also found that cell lines seemed to have relatively

high basal levels of p53 protein expression, enabling us tosuccessfully distinguish those with nonsense/frameshift/indelalterations or homozygous deletions from those with wild-typeTP53 in most cases. However, prostate tumor tissue showed lowbasal expression of p53, making it impossible to detect loss ofthe protein in intermingled tumor cells. With this limitation inmind, it will be of particular interest to determinewhether tumorswith p53 protein loss due to nonsense/frameshift/indel altera-tions or homozygous deletions, have similarly poor outcomes astumors with TP53missense mutations which are detected by ourIHC assay. If so, then it may be preferable to do NGS on prostatetumors, either alone or after IHC screening to rule out or enrichfor focal alterations. If not, then IHC alone may be a convenientand inexpensive assay that is easily deployed to communitypathology practices to test for clinically significant and heteroge-neous TP53 alterations in primary prostate tumors.

Our objective in this study was to analytically, preanalyticallyand clinically validate p53 nuclear accumulation as a prognos-tic biomarker in prostate cancer. Additional large cohorts andfuture studies are required to examine the clinical utility of p53nuclear accumulation and these studies will discern whetherthe IHC assay described herein adds substantially to currentclinical–pathologic parameters for prognostication in primaryprostate cancer. Because p53 nuclear accumulation was farmore frequent in high grade (Gleason score 9–10) carcinomascompared with all other grades, performing p53 immunostain-ing on all primary prostate cancers at diagnosis is unlikely to beefficient. Rather, we would propose that evaluation by p53 IHCbe limited to high grade, high risk tumors at diagnosis. In thisgroup, it will be important to evaluate in future studies andclinical trials whether p53 nuclear accumulation predicts dura-tion of response to hormonal therapies and/or a greater risk ofneuroendocrine transdifferentiation, adding to current prog-nostic algorithms.

Disclosure of Potential Conflicts of InterestA.E. Ross has ownership interests (including patents) in and is a consultant/

advisory board member for GenomeDx. E. Schaeffer is a consultant/advisoryboard member for GenomeDx and OPKO. T.L. Lotan reports receiving com-mercial research support from Ventana/Roche. No potential conflicts of interestwere disclosed by the other authors.

Authors' ContributionsConception and design: M.C. Haffner, A.M. De Marzo, G.J. Netto,E.M. Schaeffer, T.L. LotanDevelopment of methodology: R. Sharma, A.M. De Marzo, T.L. Lotan

Table 4. Univariable (UVA) andmultivariable (MVA)models of association of p53 and clinicopathologic risk factors for prediction of metastasis in post-biochemicalrecurrence cohort

UVA MVAVariables HR (95% CI) P HR (95% CI) P

Patient age at RP 0.99 (0.95–1.02) 0.47 0.99 (0.95–1.03) 0.55Log2 preoperative PSA (ng/mL) 1.05 (0.83–1.32) 0.68 0.97 (0.76–1.23) 0.79RP Gleason score �3þ4 Ref 1 Ref 1RP Gleason score 4þ3 2.41 (1.18–4.94) 0.02 2.43 (1.15–5.13) 0.02RP Gleason score 8 4.27 (2.33–7.81) <0.001 4.13 (2.24–7.59) <0.001RP Gleason score �9 4.84 (2.78–8.41) <0.001 3.99 (2.27–7) <0.001Positive surgical margins 0.73 (0.46–1.16) 0.18 0.71 (0.45–1.11) 0.14Extraprostatic extension 1.72 (0.93–3.18) 0.09 0.87 (0.45–1.7) 0.68Seminal vesicle invasion 2.91 (1.87–4.53) <0.001 2.2 (1.35–3.57) 0.001Lymph node invasion 1.78 (1.12–2.81) 0.01 0.95 (0.55–1.63) 0.86p53 normal Ref 1 Ref 1p53 nuclear accumulation 4.14 (2.41–7.11) <0.001 2.55 (1.1–5.91) 0.03






Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): L.B. Guedes, F. Almutairi, M.C. Haffner, A.M.DeMarzo,G.J. Netto, W.B. Isaacs, A.E. Ross, T.L. LotanAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): L.B. Guedes, G. Rajoria, Z. Liu, S. Klimek, R. Zoino,K. Yousefi, W.B. Isaacs, E.M. Schaeffer, T.L. LotanWriting, review, and/or revision of the manuscript: L.B. Guedes, K. Yousefi,G.J. Netto, A.E. Ross, E.M. Schaeffer, T.L. LotanAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): G. Rajoria, Z. Liu, T.L. LotanStudy supervision: Z. Liu, A.M. De Marzo, E.M. Schaeffer, T.L. Lotan

AcknowledgmentsThe authors thank Nicole Castagna for assistance with xenograft studies. The

authors would like to acknowledge the American Association for Cancer Research

and its financial and material support in the development of the AACR ProjectGENIE registry, as well as members of the consortium for their commitment todata sharing. Interpretations are the responsibility of study authors.

Grant SupportFunding for this research was provided in part by a Transformative Impact

Award from the CDMRP (W81XWH-12-PCRP-TIA, to T.L. Lotan) and the NIH/NCI Prostate SPORE P50CA58236.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received January 31, 2017; revised March 14, 2017; accepted April 21, 2017;published OnlineFirst April 26, 2017.

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2017;23:4693-4703. Published OnlineFirst April 26, 2017.Clin Cancer Res Liana B. Guedes, Fawaz Almutairi, Michael C. Haffner, et al.

Missense Mutation in Prostate CancerTP53Detection of Analytic, Preanalytic, and Clinical Validation of p53 IHC for

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Home | Clinical Cancer Research - Analytic, Preanalytic, and … · Prostate Cancer Liana B. Guedes1, ... as any p53 nuclear labeling in >10% of tumor cells. Fifty-four formalin-ﬁxedparafﬁnembedded(FFPE)celllinesfromtheNCI-60