pten protein loss by immunostaining: analytic validation and prognostic … · fish scoring was...

Imaging, Diagnosis, Prognosis

PTEN Protein Loss by Immunostaining: Analytic Validationand Prognostic Indicator for a High Risk Surgical Cohortof Prostate Cancer Patients

Tamara L. Lotan1, Bora Gurel1, Siobhan Sutcliffe4, David Esopi2, Wennuan Liu5, Jianfeng Xu5,Jessica L. Hicks1, Ben H. Park2, Elizabeth Humphreys3, Alan W. Partin3, Misop Han3,George J. Netto1,2,3, William B. Isaacs2,3, and Angelo M. De Marzo1,2,3

AbstractPurpose: Analytically validated assays to interrogate biomarker status in clinical samples are crucial for

personalized medicine. PTEN is a tumor suppressor commonly inactivated in prostate cancer that has been

mechanistically linked to disease aggressiveness. Though deletion of PTEN, as detected by cumbersome

FISH spot counting assays, is associated with poor prognosis, few studies have validated immunohis-

tochemistry (IHC) assays to determine whether loss of PTENprotein is associated with unfavorable disease.

Experimental Design: PTEN IHC was validated by employing formalin fixed and paraffin-embedded

isogenic human cell lines containing or lacking intact PTEN alleles. PTEN IHC was 100% sensitive and

97.8% specific for detecting genomic alterations in 58 additional cell lines. PTEN protein loss was then

assessed on 376 prostate tumor samples, and PTEN FISH or high resolution single nucleotide polymor-

phism microarray analysis was done on a subset of these cases.

Results: PTEN protein loss, as assessed as a dichotomous IHC variable, was highly reproducible,

correlated strongly with adverse pathologic features (e.g., Gleason score and pathologic stage), detected

between 75% and 86% of cases with PTEN genomic loss, and was found at times in the absence of apparent

genomic loss. In a cohort of 217 high risk surgically treated patients, PTEN protein loss was associated with

decreased time to metastasis.

Conclusion: These studies validate a simplemethod to interrogate PTEN status in clinical specimens and

support the utility of this test in future multicenter studies, clinical trials, and ultimately perhaps for routine

clinical care. Clin Cancer Res; 17(20); 6563–73. �2011 AACR.

Introduction

Prostate cancer is themost common solid organ tumor inAmerican men and the second most common cause ofcancer deaths (1). Despite improvements in early detection,we still lack molecular markers to effectively distinguishmen with high risk disease from the indolent majority.Identification of molecular aberrations that contribute tothe development of lethal disease has the additional benefitof providing specific targets for therapy. Work over the lastdecade has firmly established that loss of the PTEN tumor

suppressor (phosphatase and tensin homolog on chromo-some 10) is one of the most common somatic geneticaberrations in prostate cancer and is frequently associatedwith high risk disease (2–13). Despite these findings, how-ever, prostate cancer specimens are not yet routinely inter-rogated for PTEN loss in any clinical setting.

The majority of previous work on PTEN loss in prostatecancer has focused on genomic deletions of the PTEN locusat 10q23. Such deletions, most commonly identified byFISH, occur in 10% to 70% of prostate cancer casesdepending on the study population examined (2, 4, 6,8, 10–15) and are associated with poor prognosis (13, 16–20). Interestingly, heterozygous PTEN deletions far out-number homozygous deletions in prostate cancer and mayalso result in poor outcomes (11–13, 15–17, 20). There hasbeen much debate about whether this may be explained byhaploinsufficiency for PTEN or whether inactivation of thesecond allele in these cases has occurred but is not detect-able by FISH (21, 22). Although a number of early studiessuggested that the rate of PTEN mutation and epigeneticmodification in prostate cancer was relatively high, it isnow known that many of these initial estimates may havebeen falsely elevated due to the existence of a PTEN

Authors' Affiliations: Departments of 1Pathology, 2Oncology, and 3Urol-ogy, Johns Hopkins Medical Institutions, Baltimore, Maryland; 4Division ofPublic Health Sciences, Department of Surgery, Washington UniversitySchool of Medicine, St. Louis, Missouri; and 5Center for Genomics andPersonalized Medicine Research, Wake Forest University School of Med-icine, Winston-Salem, North Carolina

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

Corresponding Author: Angelo M. De Marzo, Cancer Research BuildingII, Room 144, 1550 Orleans Street, Baltimore, MD 21231. Phone: 410-614-9196; Fax: 410-502-9911; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-11-1244

�2011 American Association for Cancer Research.

ClinicalCancer

Research

www.aacrjournals.org 6563

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pseudogene (2, 3, 23). Recent studies have suggested thatalternative mechanisms of PTEN posttranscriptional down-regulation may play an important role in prostate cancer(24–28). To date, however, the relative frequency of PTENinactivation by mechanisms other than genomic deletionin clinical prostate cancer specimens remains unclear (29).

For clinical settings, the evaluation of allelic loss of PTENby FISH is cumbersome, requiring counting of the numberof fluorescent signals relative to control signals in partiallysectioned interphase cells. Thus, reliable detection of PTENprotein status by immunohistochemistry (IHC) in routine-ly processed clinical formalin-fixed and paraffin-embeddedpathology specimens could prove highly useful for theimplementation of PTEN status as a clinically relevantprognostic biomarker. Importantly, development of suchan assay may also detect cases where PTEN inactivationoccurs by mechanisms other than genomic deletion. Asmore therapies targeting various components of the PI3Ksignaling cascade become available, a robust assay todetermine PTEN status will likely have an important rolein clinical care. To address this need, we have developedand validated a simple and robust IHC assay to detect PTENprotein in paraffin-embedded specimens using a commer-cially available rabbit monoclonal antibody. Because ourapproach to analytical validation of PTEN staining can beported easily to other laboratories, if our findings can bevalidated in additional large cohorts, this robust IHC assaymay prove to be useful in a number of clinical arenas.

Materials and Methods

Patient and tissue selectionTen tissue microarrays (TMA) were constructed from

formalin-fixed paraffin-embedded prostate, lymph nodeor distant metastasis tissue collected from a total of 376

patients with prostate cancer who underwent radical pros-tatectomy or surgical resection of metastatic lesions at ourinstitution. Between 1 and 4 (average¼ 3) 0.6 mm cores oftumor tissue for each patient were arrayed. For mostpatients, surrounding benign prostate tissue was includedin the TMA as a control. Of these 376 patients, 56 patientswere enrolled in an adjuvant trial of docetaxel treatmentdue to high risk pathologic features (30). An additional 217patients were from a previously described cohort withbiochemical recurrence following radical prostatectomyfor clinically localized prostate cancer (31).

Finally, an additional group of 53 patients with concur-rent high resolution singlenucleotide polymorphism(SNP)microarray analysis for genomic copy number alterations intheir prostatic carcinoma were also included. Detailedresults of the genome-wide study from these patients willbe reported elsewhere (J. Xu and colleagues; unpublisheddata). For these cases, a single representative histologicsection that was adjacent to the tumor lesion harvestedfor genomic DNA was analyzed for PTEN protein loss.

Cell culture and transfectionsThree different human cell lines in which PTEN alleles

were subjected to targeted disruption using homologousrecombination were used in this study as controls for IHC,including MCF-10A mammary epithelial cells (32; gift ofK.E. Bachman), and the DLD-1 and HCT116 humancolorectal cancer cell lines (33; gift of T. Waldman).Wild-type (PTENþ/þ) heterozygous null (PTENþ/�), andhomozygous null (PTEN�/�) cell variants were maintainedas previously described (32, 33), harvested and subjectedto formalin fixation and paraffin embedding as described(34). The human prostate cancer cell lines LNCaP, DU145,and PC-3 were acquired from the American Type CultureCollection. LNCaP cells harbor one deleted allele of PTENand one mutated allele of PTEN and do not express PTENprotein (7). PC3 cells harbor a homozygous deletion ofPTEN and do not express PTENprotein (7, 10). DU145 cellsharbor an apparent coding SNP in PTEN, but are otherwisewild type and express PTEN protein (10). We also obtained56 additional cell lines of theNCI-60panel of cell lines fromthe Developmental Therapeutics Program at the NCI toevaluate PTEN staining. Cell lines were used to generatecell line microarrays (analogous to TMAs; ref. 35) using arecently updated method that will be described in detailelsewhere (D. Esopi and colleagues; unpublished data).

As an additional positive control, we obtained a mo-lecular clone of PTEN that is driven by the CMV promoter(PTEN NM_000314 Human cDNA Clone, Origene) andtransfected this construct into HCT116 PTEN�/� andDLD-1 PTEN�/� cells using Oligofectamine accordingto the manufacturer’s directions (Invitrogen). Mock orPTEN transfected cells were harvested after 48 hours andprepared for cell blocks as described above.

Fluorescence in situ hybridizationFISH was done on 2 TMAs (145 patients, 361 spots

representing a spectrum of lesions including benign

Translational Relevance

PTEN is a tumor suppressor commonly inactivated inprostate cancer. Genomic deletions of PTEN are asso-ciated with poor prognosis, but are interrogated intissue samples using relatively cumbersome FISH. Weshow that PTEN protein expression evaluated by im-munohistochemistry (IHC), using a new commerciallyavailable monoclonal antibody, is simple to conductand score after genetic validation and sensitively detectsPTEN genomic loss. Furthermore, PTEN IHC detectsadditional cases of PTEN protein loss compared withgenomic methods. Finally, loss of PTEN by IHC corre-lated with adverse pathologic features and decreasedtime to metastatic disease in a surgical cohort. Thisanalytically validated assay may be used in additionalstudies to determine its efficacy as a clinical test, includ-ing as a prognostic biomarker in needle biopsies and asa predictive marker to identify patients who couldbenefit from emerging PI3K pathway-targeted therapiesor who may be resistant to hormonal therapies.

Lotan et al.

Clin Cancer Res; 17(20) October 15, 2011 Clinical Cancer Research6564




prostate, prostatic intraepithelial neoplasia (PIN), primarytumors, and lymph node metastases) using the VysisLSI PTEN (10q23)/CEP 10 Dual Color Probe (AbbottMolecular Inc.) according to the manufacturer’s directions.Briefly, a 4 mm paraffin section was baked at 56�C for2 hours, then dewaxed and rehydrated using xylene andgraded ethanol, respectively. The TMA sections were pre-treated using Paraffin Pretreatment Reagent Kit III (AbbottMolecular Inc.). TMAs and the PTEN/CEP10 FISH probeswere codenatured at 94�C for 5 minutes and hybridizedovernight at 37�C in a humid chamber (StatSpin Thermo-Brite; IRIS Inc.).

FISH scoringFISH scoring was conducted using a Nikon 50i

epifluorescence microscope equipped with X-Cite series120 illuminator (EXFO Photonics Solutions Inc.) and a100�/1.4 NA oil immersion Neofluar lens. Grayscaleimages were captured for presentation using NikonNIS-Elements software and an attached PhotometricsCoolsnapEZ digital camera, pseudo-colored and merged.

FISH interpretationFISH interpretation was done by a pathologist (B.G.)

blinded to the IHC results. To establish normal cutoffs forscoring and to ensure that FISH signal truncation seen intissue sections was not scored as a false positive, the PTEN/centromere 10 (CEP10) signals were tallied in 50 nonneo-plastic normal-appearing prostate specimens contained onthe same TMAs as the experimental specimens, and themean and SD for PTEN/CEP10 ratios and percentage ofepithelial cells containing 0 or 1 PTEN probe were calcu-lated. After scoring 30 cells, a tumor was considered to havePTEN loss if the percentage of cells showing 0 or 1 PTENprobe signal was greater than 2 SD above the mean estab-lished from the normal tissue and the PTEN/CEP10 ratiowas more than 2 SD below the mean established in normaltissue. A case that was represented by more than 1 tissuecore on the TMA was considered to have PTEN loss if anyTMA spot in the case showed PTEN loss.

ImmunohistochemistryA number of commercial antibodies were evaluated to

identify those most highly sensitive and specific in IHC cellline staining experiments. These antibodies included thefollowing: Zymed/Invitrogen rabbit polyclonal catalogNo. 51-2400; Epitomics rabbit monoclonal clone Y184;Cascade BioSciences mouse monoclonal clone 6H2.1;Santa Cruz Biotechnologies mouse monoclonal clone28H6; and Cell Signaling Technology rabbit monoclonalD4.3. Using the PTEN positive and negative cell linesdescribed above, we found that the rabbit monoclonalantibody from Cell Signaling Technology performed thebest overall at the following conditions: Antigen unmask-ing was done by steaming in EDTA buffer (pH 8.0) for45 minutes. Endogenous peroxidase activity wasquenched by incubation with peroxidase block for 5 min-utes at room temperature. Nonspecific binding was

blocked by incubating in 1% bovine serum albumin inTris-HCl pH 7.5 for 20 minutes at room temperature.Slides were incubated with a 1:100 dilution of rabbitmonoclonal anti-PTEN antibody (clone D4.3, #9188, CellSignaling Technologies) overnight at 4�C. A horseradishperoxidase-labeled polymer (PowerVision Poly-HRP anti-Rabbit IgG; Leica Microsystems) was then applied for30 minutes at room temperature. Signal detection wasdone using 3,30-diaminobenzidine tetrahydrochloride(DAB) as the chromagen. Slides were counterstained withhematoxylin, dehydrated, and mounted. In a number ofexperiments, we also evaluated another rabbit monoclonalantibody from Cell Signaling Technology (clone 138G6)and found it to perform similarly to the D4.3 clone.

Interpretation of IHCPTEN protein was visually scored using a dichotomous

scoring system by 2 urologic pathologists (T.L.L. andA.M.D.). IHC scoring was blinded with respect to FISHand SNP array results, pathologic stage, and final Gleasonscore at radical prostatectomy, as well as patient outcome.Using this system, each spot of tumor tissue was scored asnegative or positive for PTEN protein by comparing stain-ing inmalignant glands with that of adjacent benign glandsand/or stroma which provided an internal positive controlwithin each tissue core. Staining was classified as negativeif the intensity was markedly decreased or entirely negativeacross all tumor cells compared with the surroundingbenign glands and/or stroma. A given spot was droppedfrom the analysis if these benign areas lacked PTEN staining(this occurred in <5 TMA spots total).

High resolution SNP microarray analysisPTEN IHCwas done on an additional 53 prostate tumors

for which PTEN copy number data was available from highresolution SNP microarray analysis (Affymetrix 500 K).Further details regarding the genome wide copy numberanalyses will be provided in a separate publication (J. Xuand colleagues; unpublished data).

Statistical analysisPearson c2 and Fisher’s exact tests were used to determine

the association of PTEN protein loss with pathologic vari-ables and PTEN genomic loss. To investigate univariableassociations between PTEN loss and time to distant me-tastasis and prostate cancer-specific death, Kaplan–Meiersurvival curves were calculated and compared by log-ranktests. Multivariable-adjusted associations were investigatedusing Cox proportional hazards regression; models includ-ed terms for pathologic stage, Gleason grade, preoperativePSA, and surgical margin status. Statistical analyses weredone using SAS version 9.1.

Results

Validation of IHC assayWe first validated our IHC assay analytically for PTEN

protein loss using formalin fixed paraffin-embedded

PTEN Protein Loss in Prostate Cancer

www.aacrjournals.org Clin Cancer Res; 17(20) October 15, 2011 6565




cell blocks created from 3 distinct isogenic human celllines with and without PTEN deletion. In wild-type HCT-116, MCF-10A, and DLD-1 cells, a variable degree ofcytoplasmic and nuclear PTEN protein immunostainingwas evident, whereas isogenic lines which had undergone2 rounds of somatic homologous recombination (result-ing in disruption of both alleles of PTEN) showedcomplete absence of PTEN protein by IHC (Fig. 1). InMCF-10A cells that were heterozygous for PTEN disrup-tion (PTENþ/�), there were intermediate levels of stainingbetween wild-type (PTENþ/þ) and homozygous mutantvariants (PTEN�/�; Fig. 1). In contrast, PTEN�/� HCT-116or DLD-1 cells transiently transfected with an expressionvector containing a cDNA encoding full-length humanPTEN showed anumber of cells with greatly increased PTENprotein by IHC (Fig. 1). Taken together, these isogenic cellline experiments verify the specificity of the Cell SignalingTechnologies rabbit monoclonal antibody and our IHCprotocol. For additional validation, we also stained cellpellets that were obtained from PC3, LNCaP, and DU145human prostate cancer cell lines that have known PTENgenomic status. As expected, bothPC3cells andLNCaPwerecompletely negative for staining, whereas DU145 cells

showed moderate levels of both cytoplasmic and nuclearstaining for PTEN (not shown). To further assess the per-formance of the assaywenext examined55of the remainingcell lines from the NCI-60 cell line panel, because PTENgenomic status has been reported from these lines (Sup-plementary Table S1; 36, 37). The sensitivity for IHC stain-ing to identify cell lineswithpredictedoncogenic alterationsin PTEN was 100% (13 of 13 lines with mutant PTENshowed loss of IHC staining), with a specificity of 97.8%(44 of 45 cell lines without PTEN mutation did not showPTEN protein loss by IHC).

PTEN protein scoring in human prostate tissuesPTEN protein loss by IHC was scored on paraffin-em-

bedded prostate tissues from a total of 376 patients on10 TMAs. PTEN protein was expressed in all (100%) casesof benign prostate tissue present on TMAs. Although therewas some variability in staining intensity, PTEN proteinwas diffusely expressed in the cytoplasm and nucleus ofluminal and basal epithelial cells, as well as in the sur-rounding stromal tissue (Fig. 2). In fact, virtually all celltypes present (including endothelial cells, smooth musclecells, inflammatory cells, and peripheral nerves) were

A B C

D E F

G H I

Figure 1. PTEN proteinexpression by IHC in isogenic cellline controls with and withoutsomatic PTEN gene loss. A, wild-type HCT116 and (D) DLD-1 coloncancer cells, and (G) MCF-10Abreast epithelial cells, show PTENprotein expression by IHC,whereas the same cell lines withhomozygous PTEN deletionby somatic homologousrecombination (PTEN KO) showabsent PTEN protein (B, E, H).C and F, HCT116 and DLD-1 cellswith PTEN deletion transientlytransfected with CMV-PTEN showhigh PTEN protein expression in asubset of cells. I, MCF10A cellswith hemizygous deletion ofPTEN show levels of PTENimmunostaining intermediatebetween wild type and cells withhomozygous PTEN deletion.

Lotan et al.





positive for PTEN staining. This pattern of staining was alsofound on 53 standard tissue slides (see below).To score PTEN protein expression in prostatic carcinoma,

we devised a binary scoring system wherein malignantglands were scored as positive or negative for cytoplasmicPTEN protein relative to the internal control of surround-ing benign glands and/or stroma (Fig. 2). We consideredcases in which the majority (>90%) of cells retained easilydiscernible levels of PTEN staining to be positive for PTENprotein (e.g., Fig. 2C, left panel). Cases considered negativefor PTEN protein either showed a complete absence ofPTEN staining or extremely weak intensity staining in morethan 10% of cells. In the latter cases, however, the overallstaining was always strikingly reduced as compared withsurrounding tissue, particularly in nuclei which appearedblue only without discernible brown staining. Interobserv-er reproducibility for this simple scoring system was high.On an array with 117 tumor spots scored independently ina blinded fashion by 2 pathologists (T.L.L. and A.M.D.),there was 95% (111/117 spots) agreement on the presenceor absence of PTEN protein by IHC in malignant glands(k ¼ 0.87 or "almost perfect" agreement). In this set, 26%to 27% (depending on the observer) of the spots showedPTEN loss in tumor cells.A fraction of tumors arrayed on the TMAs showed

prominent intratumoral heterogeneity for PTEN expres-sion, with some spots positive for PTEN whereas other

spots from the same index tumor were negative. Overall,intratumoral heterogeneity in PTEN expression was ob-served in 9% (20/220 cases) of primary tumors. Occasion-ally, this heterogeneity of PTEN protein expression wasseen even within a given TMA spot, with some malignantglands expressing PTEN whereas adjacent glands (or evenadjacent cells within a malignant gland) were entirelynegative (Fig. 2D and E). For the purposes of this study,a case was scored as PTEN negative if any tumor spot fromthat case showed greater than 10% of tumor cells withmarkedly decreased PTEN protein. Similarly, a total of 53prostatic tumors were scored for PTEN expression usingsingle tumor sections on standard histologic slides ratherthan TMAs, and 11% (6/53) of these cases showed 2 clearpopulations of tumor cells where one population hadPTEN loss and the other did not. In this setting, as above,a case with PTEN protein loss in greater than 10% of thesectioned tumor was considered to have PTEN loss for thepurposes of data analysis.

Association of PTEN protein loss with pathologicvariables and tissue type

Overall, PTEN protein was scored by IHC in 397 tissuesamples collected from 376 individual patients. Pathologicstage data was available on 263 patients (66%) andGleason grade was available on all tissues. In these samples,PTEN protein loss by IHC was highly correlated with

A B C

D E F

Figure 2. PTEN protein expression by IHC in human prostate specimens. A, typical cancer case with uniform retained PTEN protein expression in allmalignant glands. B, typical cancer case with PTEN protein loss in malignant glands, whereas adjacent benign glands (arrow) retain PTEN protein expression.C, some tumors showed only weak PTEN protein positivity (left) but were still readily distinguishable from cases with total PTEN protein loss (right). D,intratumoral heterogeneity for PTEN protein expression is evident in this tumor specimen, where malignant glands with PTEN loss and PTEN proteinretention are intermingled. Note the presence of cells with and without PTEN expression within a single malignant gland (arrow). E, high grade PIN withPTEN loss in most of the involved gland, whereas PTEN protein is retained in a minority of luminal cells (arrow) and all basal cells (arrowhead). An adjacentbenign gland expresses PTEN. F, PTEN protein loss in lymph node metastasis of prostatic carcinoma. Although the cytoplasm is negative, some glandsshow a small amount of staining at the apical plasma membrane (arrow), a finding of uncertain significance.






increased pathologic stage (P ¼ 0.003) and Gleason grade(P < 0.0001, Fig. 3). Overall, 41% (17/41) of pT3bN0 casesshowed PTEN loss, compared with 34% (39/112) ofpT3aN0 cases and 14% (6/44) of pT2N0 cases. Similarly,45% (65/144) of Gleason 8 to 10 cases showed PTENprotein loss compared with 39% (61/155) of Gleason 7cases and 20% (20/98) of Gleason 5 to 6 cases.

Consistent with the correlation with pathologic stageand grade, PTEN loss by IHC was also highly correlatedwith the tissue diagnosis (Fig. 3). High grade PIN showedthe lowest rate of PTEN loss (12% or 3/25 cases). Incontrast, pelvic lymph node metastases and distantmetastases showed the highest rates of PTEN loss (46%or 20/43 cases and 57% or 4/7 cases, respectively). Primaryprostatic tumors showed an intermediate rate of PTENloss when considered as a group (38% or 119/308).

Sensitivity of PTEN IHC for detecting PTEN genomicloss

To determine whether the PTEN IHC assay was sensitiveor specific for detecting PTEN allelic loss in clinical prostatecancer specimens, we evaluated PTEN protein by IHC in aseries of prostate tumors for which we also had available

PTEN genomic status by concurrent FISH (66 cases, ofwhich 56 were from the high risk group enrolled in anadjuvant trial of docetaxel) or high resolution copy numberSNP microarray analysis (53 cases). For the cases withconcurrent PTEN FISH data, presence or absence of PTENprotein was highly correlated with presence or absence ofPTEN loss of heterozygosity (Fig. 4, Table 1, P ¼ 0.0044 byFisher’s exact test). Of 66 primary prostate tumors withavailable PTEN FISH data, 24 (36%) had loss of at least 1PTEN allele. Of these, 75% (18/24) had loss of PTENprotein by IHC, indicating that PTEN IHC is sensitivefor the detection of genomic PTEN loss. For the cases withconcurrent SNP microarray copy number analysis, PTENIHC was also highly correlated with PTEN genomic status(Table 2, P ¼ 0.00026 by Fisher’s exact test). Of 53 caseswith SNPmicroarray data, 41% (22 cases) showed loss of atleast 1 allele of PTEN. Of these cases, 86% (19/22) showedPTEN protein loss by IHC. Of the cases with SNP micro-array data and heterogeneous intratumoral PTEN IHCstaining, 33% (2/6 cases) showed no evidence of genomicPTEN loss, 50% (3/6 cases) showed loss of one PTEN allele,and 17% (1/6) showed homozygous loss of both PTENalleles.

A PTEN protein loss and pathologic stage

% w

ith P

TE

N loss b

y IH

C

% w

ith P

TE

N loss b

y IH

C

% w

ith P

TE

N loss b

y IH

C

T2N0

Benign PIN Primarytumors

Lymph nodemetastases

Distantmetastases

P = 0.003

n = 66n = 41

n = 112

n = 98

n = 155n = 144

n = 308

n = 25n = 14

n = 43

n = 7

n = 44

P = 0.0001

P = 0.001

100

80

60

40

20

100

80

60

40

20

100

80

60

40

20

T3aN0 T3bN0 TXN1 ≤6 7 8–10

PTEN protein loss and specimen type

PTEN protein loss and Gleason gradeB

C

Figure 3. PTEN protein loss byIHC is highly correlated withprostate cancer pathologic stageand grade. A, PTEN protein ismore frequent in higher pathologicstage tumors (P ¼ 0.003 byPearson's c2 test). B, PTENprotein loss is more commonin higher Gleason grade tumors(P ¼ 0.0001 by Pearson's c2 test).C, PTEN protein loss is leastcommon in benign prostatetissues and PIN and mostcommon in metastatic prostatetumors (P¼ 0.001 by Pearson's c2

test).

Lotan et al.





PTEN IHC was as sensitive for the detection of cases withloss of one PTEN allele (heterozygous deletion) as it was forthe detection of cases with loss of both alleles (homozy-gous deletion; 87% or 13/15 vs. 86% or 6/7, respectively,Table 3). Importantly, of cases with PTEN protein loss byIHC, 45% (15/33) and 37% (11/30) did not have anyPTEN genomic loss detectable by FISH or SNP microarray,respectively.

Correlation of PTEN protein loss with clinicaloutcomeA subset of the RRP specimens are part of a previously

described retrospectively studied high risk surgical cohortof patients who underwent radical prostatectomy (RRP) by

a single surgeon at the Johns Hopkins Hospital between1986 and 1996 and subsequently developed biochemicalrecurrence (Ref. 31; Table 3). Of note, none receivedneoadjuvant chemotherapy or hormonal therapy priorto development of metastatic disease (31). Median patientfollow-up was 16 years and tissue was present for TMAconstruction from 217 of the original 304 cases in thiscohort. At RRP, 22% (49/217) had a Gleason score (GS) of6 or less, 51% (110/217) had GS of 7 and 27% (58/217)had a GS of 8 to 10. 83% (181/217) had extracapsularextension, 34% (74/217) had seminal vesicle involvementand 27% (58/217) had lymph node metastases. 36%(78/217) had positive surgical margins. By definition inthis cohort, 100% (217/217) of patients had biochemicalrecurrence during the follow-up period, with 25%(52/210) showing evidence of local recurrence and 60%(124/208) with distant metastases during follow-up.38% (79/207) of patients died of prostate cancer duringthe study period. In Kaplan–Meier analysis, loss of PTENimmunostaining was significantly associated with de-creased time to metastasis (P ¼ 0.03, Fig. 5). There wasa correlation between PTEN protein loss and decreasedtime to prostate-specific death, although this did not reachstatistical significance (P ¼ 0.06). In multivariable Coxregression analysis, PTEN protein was not associated withtime to metastasis or prostate cancer death (Table 3).

Discussion

PTEN genomic loss was first identified as a molecularaberration common in prostate cancer nearly 15 years ago(2, 37). In early studies using microsatellite analysis, LOHat the PTEN locus was reported in 10% to 55% of primarytumors from surgical cohorts (2, 4–6, 8, 9). In more recentstudies using FISH, loss of at least 1 PTEN allele has beenreported in as few as 17% of patients with tumors inci-dentally discovered on transurethral resection (TURP),however PTEN allelic loss is present in 17% to 68% ofprimary tumors from surgical cohorts and up to 77% ofhormone-resistant primaries discovered on TURP (11–13,15–17, 20, 38). In general, PTEN loss is more common inprostate cancer metastases than in primary tumors, withrates of loss reported near 50% in 3 independent studies

CASE A

PTEN/CEP10 = 0.84 PTEN/CEP10 = 0.18

PT

EN

IH

CP

TE

N F

ISH

CASE B

Figure 4. PTEN protein expression by IHC is highly correlated with PTENgenomic loss by FISH. In case A, PTEN protein is highly expressed inmalignant glands, corresponding to a normal PTEN/ CEP10 ratio by FISH,with retention of PTEN (red) and CEP10 (green) FISH signals in malignantcells. In case B, PTEN protein is markedly decreased in malignant glands,with a corresponding PTEN/CEP10 ratio of 0.18. Malignant cells showhomozygous loss of the PTEN (red) signal in malignant cells, with retentionof the CEP10 (green signal).

Table 1.Correlation between PTEN protein lossdetected by IHC and PTEN genomic lossdetected by FISH

PTEN genomic loss by FISH PTEN protein by IHC

Present Absent

Normal 27 15Deleted 6 18

NOTE: P ¼ 0.0044 by Fisher's exact test.

Table 2.Correlation between PTEN protein lossdetected by IHC and PTEN genomic copy num-ber change detected by SNP array

PTEN genomic lossby SNP array

PTEN protein by IHC

Present Absent

No change 20 11Heterozygous 2 13Homozygous 1 6

NOTE: P ¼ 0.00026 by Fisher's exact test.






using different methods of detection (6, 15, 19). Despitethe variations in reported rates of genomic PTEN loss, anearly universal finding in the FISH studies is that loss of 1PTEN allele is significantly more frequent than loss of bothPTEN alleles in surgical cohorts. However, rates of homo-zygous loss also vary by the cohort examined. Althoughhomozygous loss of PTEN hovers around 0% to 10% ofprimaries, it is closer to 50% of metastatic or hormone-resistant cases (11–13, 15–17, 20).

Overall, the wide range in reported frequency of PTENgenomic loss in prostate cancer likely reflects the closeassociation of PTEN loss with high risk pathologic fea-tures and possibly an association with androgen-insensi-tive disease. Thus, the frequency of PTEN loss is higher incohorts enriched for aggressive disease, with increasedpathologic stage and Gleason grade (18). Despite theclose association with pathologic variables, at least 2separate studies have found that PTEN genomic loss isindependently correlated with decreased time to bio-chemical recurrence (13, 16, 20). However, at least 3separate studies done in TURP and surgical cohorts havenot found an association between PTEN genomic loss andsurvival (16–18).

Interestingly, the association between PTEN genomicloss and biochemical recurrence has most commonly beendocumented for hemizygous deletions, likely because theyare much more frequent than homozygous deletions. Al-though several studies have shown higher HR for homo-zygous compared with hemizygous PTEN loss, the

correlation between loss of only 1 allele of PTEN anddecreased time to biochemical recurrence remains signifi-cant (13, 16, 20). This suggests that either PTEN is ahaploinsufficient gene, or in cases of hemizygous loss,the second allele is commonly inactivated by additionalmechanisms which are not detected by FISH. Althoughthere is some evidence for PTEN haploinsufficiency in themouse, data to support this hypothesis are lacking inhumans (21, 22). Thus the correlation between loss of asingle PTEN allele and features of aggressive disease inprostate cancer strongly suggests that alternative epigeneticor perhaps nongenomic mechanisms of PTEN inactivationplay an important role in prostate cancer progression (29).This also suggests that FISH may be systematically under-estimating the frequency of PTEN loss in prostate cancer.

The possibility that PTEN FISH may fail to detect somecases of prostate cancer with PTEN inactivation stronglyargues for the need for an alternative assay to detect PTENloss. Although IHC to detect PTEN protein levels is anobvious alternative, until recently, studies using thismethod have been impeded by the lack of reliable anti-bodies and by a paucity of dependable genetic controls.To our knowledge, there are at least 10 prior studies in theliterature looking at the utility of PTEN IHC for thedetection of PTEN loss in prostate cancer (10–12, 14–16, 39–42). Although some found an association betweenPTEN protein loss and Gleason grade, stage, or biochem-ical recurrence, many did not. Some of this variation maybe due to antibody performance. While this manuscript

Table 3. HR and 95% confidence intervals (CI) of distant metastasis and disease-specific death by PTENloss and clinico-pathologic characteristics

Variable Number(%)

Unadjusted HR(95% CI) ofdistantmetastasis

Multivariable-adjusted HR(95% CI) of distantmetastasis

UnadjustedHR (95% CI) ofdisease-specificdeath

Multivariable-adjusted HR(95% CI) ofdisease-specificdeath

PTEN protein Present 62 (29) 1 1 1 1Absent 155 (71) 1.49 (1.02–2.16) 1.13 (0.77–1.67) 1.54 (0.97–2.44) 1.18 (0.72–1.92)

Gleason score 6 49 (22) 1 17 110 (51) 1.85 (.99–3.47) 3.53 (1.36–9.17)8–10 58 (27) 4.51 (2.27–8.96) 10.8 (3.97–29.1)

Pathologic stage pT2 61 (28) 1 1pT3a 82 (38) 1.63 (0.72–3.71) 1.35 (0.46–3.99)pT3b 74 (34) 2.68 (1.10–6.48) 2.42 (0.77–7.63)

Lymph nodes Negative 159 (73) 1 1Positive 58 (27) 5.54 (2.34–13.1) 4.03 (1.35–12.1)

Surgical margins Negative 139 (64) 1 1Positive 78 (36) 0.77 (0.52–1.14) 1.01 (0.62–1.66)

Biochemical recurrence 217 (100)Local recurrence Negative 158 (75)

Positive 52 (25)Distant metastasis Negative 84 (40)

Positive 124 (60)Disease-specific death Negative 128 (62)

Positive 79 (38)

Lotan et al.





was in preparation, a study evaluating commerciallyavailable PTEN antibodies for IHC found that manyantibodies resulted in nonspecific nucleolar staining incell lines with known PTEN genomic loss (43). Impor-tantly, this study independently corroborated our find-ings that recently available rabbit monoclonal antibodiesto PTEN performmuch more reliably than older clones orpolyclonal antibodies. In addition to problems with olderantibodies, earlier studies also employed widely variableand often complex scoring systems for PTEN protein, anddid not account for interobserver variability in scoring.Only 2 studies have looked at the association betweenPTEN protein loss and disease progression and survival inprostate cancer, and whereas 1 study scored cytoplasmicstaining intensity and extent, the other found an associ-ation only with nuclear PTEN staining (14, 16).Given the wide variation of methodology and results in

the literature, we set out to develop an IHC assay for PTENthat would be simple enough to allow routine use inclinical pathology specimens. One advantage of our studyis that we had access to a number of isogenic cell lines bothwith and without PTEN genomic deletion. Using these celllines, we optimized a staining protocol for a recentlyavailable rabbit monoclonal antibody, wherein all lineswith PTEN deletion showed a complete absence of PTEN

protein. With our staining protocol, background benignprostatic glands and stroma showed robust staining forPTEN. Because of the presence of an internal positivecontrol in all samples, we were able to apply a simpledichotomous and highly reproducible scoring system formalignant glands, with cytoplasmic PTEN either present, ormarkedly decreased. Interestingly, Sangale and colleaguesalso applied a simple dichotomous scoring strategy in theirapproach to PTEN IHC scoring in clinical samples (43).

Perhaps one of the most important advantages of ourstudy is that we were able to correlate PTEN proteinexpression with PTEN genomic status. We found that PTENIHC is highly sensitive for detection of PTEN genomic loss,detecting nearly 80% of cases with loss by FISH and morethan 80% of cases with loss by high resolution SNP array.Only 3 previous studies have validated their IHC assay in asimilarly rigorous fashion. Yoshimoto and colleaguesreported PTEN levels as a product of the intensity andpercentage of cytoplasmic and/or nuclear staining andalthough they did not provide data for each individualcase, they found an overall correlation between PTENprotein levels as a continuous variable and PTEN genomicstatus (12). Verhagen and colleagues employed a similarscoring system and found that 66% (10/15) of cases withPTEN deletion by FISH showed PTEN protein loss (11).Recently, Han and colleagues did a similar study wherethey scored cytoplasmic staining intensity on a 0-2þ scaleand correlated with FISH results (15). They reported thatthey detected 52% of cases with PTEN loss by FISH usingIHC if only cases with 0þ immunostaining were consid-ered to be truly PTEN protein negative.

One of the most intriguing findings in the current studywas that 45% and 37% of tumors with PTEN protein lossdid not show genomic deletions detectable by FISH or highresolution SNP microarray, respectively. Although it ispossible that FISH may not detect some small deletionsin PTEN, the similar data obtained from the high resolu-tion SNP microarray suggests that this is a less likelyexplanation for our findings. In addition, we found thatPTEN IHC was as sensitive for the detection of hemizygousloss by SNP array as it was for the detection of homozygousloss. Although even high resolution SNP microarrayscan miss very small deletions depending on a numberof factors, this data strongly suggest that in addition togenomic deletion, alternative mechanisms for PTEN inac-tivation likely exist. Interestingly, other authors havereported similar findings. Han and colleagues reported that35% (6/17) of their PTEN protein negative cases did notshow genomic deletion by FISH and Verhagen and collea-gues reported that 33% (5/15) cases without PTEN proteinshowed no evidence of deletions by FISH (11, 15).

The frequency with which PTEN is inactivated by muta-tions, epigenetic modifications and/or nongenomic meansremains unclear in prostate cancer. Although early studiesreported a high rate of mutations and methylation in thePTEN promoter region, it is likely that some of theseestimates were falsely elevated because of detection of aPTEN pseudogene that harbors a high rate of such changes

A Time of distant metastasis

Time of prostate cancer-specific death

Time (y)

Time (y)

5 10 15 20 25 30

5 10 15 20 25 30

% m

eta

sta

sis

fre

eD

iseas

e-s

pe

cif

ic s

urv

iva

l

100%

75%

50%

25%

100%

75%

50%

25%

PTEN normalPTEN protein loss

P = 0.03

P = 0.06

B

Figure 5. PTEN protein loss by IHC is associated with poor clinicaloutcomes in a surgical cohort of high risk prostate cancer patients. A, theKaplan–Meier curve shows a significant decrease in metastasis-freesurvival for patients with PTEN protein loss by IHC (P ¼ 0.03). B, theKaplan–Meier curve for disease-specific survival shows a nonsignificantdecrease in prostate-cancer specific survival in patients with PTEN proteinloss (P ¼ 0.06).






(2, 3, 11, 15, 23, 37, 44–46). More recent studies havedocumented only rare cases in which PTEN is inactivatedby point mutations or small insertions or deletions (indels;refs. 19, 28, 47). In addition, the functional consequencesof PTEN promoter methylation for PTEN expression areunclear because recent findings that the PTEN promoteris sharedwith that of a p53-target gene (KILLIN)whichmaybe a tumor suppressor in its own right (48). Recent studieshave elucidated the role of microRNAs and pseudogenedeletion in the regulation of PTEN protein levels (26, 27).Furthermore, a separate study identified chromosomaltranslocations in MAGI-2, a membrane-associated guany-late kinase known to bind and stabilize PTEN protein (28,49). Overall, these data strongly suggest that PTEN inacti-vation in prostate cancer occurs through a number ofmechanisms, many of which have yet to be described. This,in turn, highlights the importanceof an assay thatwill detectPTEN inactivation occurring via multiple mechanisms.

Although several studies have shown an associationbetween PTEN protein expression and the surrogate clinicalendpoint of biochemical recurrence, only 2 prior studieshave examined whether PTEN protein expression isassociated with metastasis and death in prostate cancer.Halvorsen and colleagues looked at PTEN protein expres-sion in prostate tumors from 104 surgically treated patientsand found that cytoplasmic PTEN expression was an in-dependent predictor of locoregional recurrence in thiscohort (14). McCall and colleagues found that nuclearPTEN was independently associated with disease specificsurvival in a group of 68 tumors in TURP specimens (16).In our cohort of 217 patients with biochemical recurrence,we found that PTEN protein expression, as a single variable,was a significant predictor of decreased time to metastasisand correlated with decreased time to prostate cancer–specific death (although this did not reach statistical sig-nificance). Given that this cohort was significantly enrichedfor high risk pathologic features and that PTEN protein lossis highly correlated with such features, it is likely that PTENprotein expression as a single variable prognosticator willperform even better in more typical surgical cohorts.

Ultimately, the IHC test for PTEN protein expressiondescribed herein will most likely be of use in the setting ofprostate tumors diagnosed on needle biopsy for a numberof reasons. First, this simple test, which is easier andcheaper to conduct than FISH, could easily be routinelydone on needle biopsy specimens and compared withFISH, will likely identify additional cases lacking PTENprotein. Second, although we found PTEN protein losswas highly correlated with tumor grade and pathologicstage in radical prostatectomy cases (and thus not inde-pendently associated with clinical outcome), in the set-ting of needle biopsies, pathologic tumor stage isunknown and tumor grade is routinely underestimatedin approximately 20% of cases. In this way, as a prog-nostic biomarker, loss of PTEN protein in a needle biopsyspecimen may be useful for the identification of pre-sumed low risk prostate cancer patients (such those onactive surveillance) that are prone to disease progressionand hence require treatment. Third, as a predictive bio-marker, loss of PTENmay prove useful for the selection ofappropriate patients for treatment with emerging PI 3-Kinase pathway-targeted therapies, a number of which arecurrently in clinical trials for prostate cancer. Finally,PTEN loss may also serve as a biomarker of hormonaltherapy resistance in advanced prostate cancer (50).

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Grant Support

This work was funded in part by the NIH/NCI Prostate SPORE P50CA58236and the Patrick C. Walsh Prostate Cancer Research Fund of which A.M. DeMarzo is the Peter J. Sharp Scholar.

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 May 13, 2011; revised July 27, 2011; accepted August 7, 2011;published OnlineFirst August 30, 2011.

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2011;17:6563-6573. Published OnlineFirst August 30, 2011.Clin Cancer Res Tamara L. Lotan, Bora Gurel, Siobhan Sutcliffe, et al. Cancer PatientsPrognostic Indicator for a High Risk Surgical Cohort of Prostate PTEN Protein Loss by Immunostaining: Analytic Validation and

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