Genomic Deletion of PTEN Is Associated with Tumor Progression and Early PSA Recurrence in ERG Fusion-Positive and Fusion-Negative Prostate Cancer

      The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) gene is often altered in prostate cancer. To determine the prevalence and clinical significance of the different mechanisms of PTEN inactivation, we analyzed PTEN deletions in TMAs containing 4699 hormone-naïve and 57 hormone-refractory prostate cancers using fluorescence in situ hybridization analysis. PTEN mutations and methylation were analyzed in subsets of 149 and 34 tumors, respectively. PTEN deletions were present in 20.2% (458/2266) of prostate cancers, including 8.1% heterozygous and 12.1% homozygous deletions, and were linked to advanced tumor stage (P < 0.0001), high Gleason grade (P < 0.0001), presence of lymph node metastasis (P = 0.0002), hormone-refractory disease (P < 0.0001), presence of ERG gene fusion (P < 0.0001), and nuclear p53 accumulation (P < 0.0001). PTEN deletions were also associated with early prostate-specific antigen recurrence in univariate (P < 0.0001) and multivariate (P = 0.0158) analyses. The prognostic impact of PTEN deletion was seen in both ERG fusion-positive and ERG fusion-negative tumors. PTEN mutations were found in 4 (12.9%) of 31 cancers with heterozygous PTEN deletions but in only 1 (2%) of 59 cancers without PTEN deletion (P = 0.027). Aberrant PTEN promoter methylation was not detected in 34 tumors. The results of this study demonstrate that biallelic PTEN inactivation, by either homozygous deletion or deletion of one allele and mutation of the other, occurs in most PTEN-defective cancers and characterizes a particularly aggressive subset of metastatic and hormone-refractory prostate cancers.
      Prostate cancer is a leading cause of cancer-related mortality in men. More than 600,000 men are annually diagnosed as having prostate cancer worldwide.
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      Although most prostate cancers are detected at early stages as a result of prostate-specific antigen (PSA) screening, many patients harbor advanced and metastatic cancer at diagnosis. A better understanding of the molecular biological features of prostate cancer may help to improve prostate cancer diagnosis and therapy.
      Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) was identified as a tumor suppressor gene on chromosome 10q23
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      Prostate-specific deletion of the murine Pten tumor suppressor gene leads to metastatic prostate cancer.
      and it has been discussed whether a reduced PTEN gene dosage (haploinsufficiency) might be sufficient to cause prostate cancer.
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      Pten dose dictates cancer progression in the prostate.
      Moreover, two recent studies
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      Aberrant ERG expression cooperates with loss of PTEN to promote cancer progression in the prostate.
      suggested a cooperative effect between PTEN inactivation and ERG fusion in prostate cancer initiation and progression.
      Although the importance of PTEN inactivation for prostate cancer biological features is undisputed, data on the prevalence and prognostic relevance of PTEN alterations in clinical prostate cancer specimens are inconsistent.
      • McMenamin M.E.
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      Loss of PTEN expression in paraffin-embedded primary prostate cancer correlates with high Gleason score and advanced stage.
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      Loss of PTEN is associated with progression to androgen independence.
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      Determining risk of biochemical recurrence in prostate cancer by immunohistochemical detection of PTEN expression and Akt activation.
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      Allelic losses at loci on chromosome 10 are associated with metastasis and progression of human prostate cancer.
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      Distinct areas of allelic loss on chromosomal regions 10p and 10q in human prostate cancer.
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      Frequent inactivation of PTEN/MMAC1 in primary prostate cancer.
      Only a few studies have analyzed the frequency of PTEN deletions using fluorescence in situ hybridization (FISH) analysis, which is regarded as the gold standard for determination of gene copy numbers in tissue samples, or performed sequence analysis to estimate the prevalence of PTEN mutations. In these studies, PTEN deletions were reported from 17% to 68%
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      Fluorescence in situ hybridization study shows association of PTEN deletion with ERG rearrangement during prostate cancer progression.
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      Interphase FISH analysis of PTEN in histologic sections shows genomic deletions in 68% of primary prostate cancer and 23% of high-grade prostatic intra-epithelial neoplasias.
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      Absence of TMPRSS2: ERG fusions and PTEN losses in prostate cancer is associated with a favorable outcome.
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      Is PTEN loss associated with clinical outcome measures in human prostate cancer?.
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      • Squire J.A.
      PTEN genomic deletion is associated with p-Akt and AR signalling in poorer outcome, hormone refractory prostate cancer.
      and PTEN mutations were found in up to 21% of prostate cancers.
      • Cairns P.
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      • Halachmi S.
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      • Herman J.G.
      • Jen J.
      • Isaacs W.B.
      • Bova G.S.
      • Sidransky D.
      Frequent inactivation of PTEN/MMAC1 in primary prostate cancer.
      • Suzuki H.
      • Freije D.
      • Nusskern D.R.
      • Okami K.
      • Cairns P.
      • Sidransky D.
      • Isaacs W.B.
      • Bova G.S.
      Interfocal heterogeneity of PTEN/MMAC1 gene alterations in multiple metastatic prostate cancer tissues.
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      • Heise C.
      • McClintock D.E.
      • Grant C.D.
      • Chung L.W.
      • Frierson Jr, H.F.
      PTEN/MMAC1 is infrequently mutated in pT2 and pT3 carcinomas of the prostate.
      • Pourmand G.
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      • Saadati H.R.
      Role of PTEN gene in progression of prostate cancer.
      To comprehensively study the prevalence and potential clinical significance of PTEN deletions and their relationship to PTEN mutations and methylation, we took advantage of a pre-existing TMA containing >4000 prostate cancers with clinical follow-up data.

      Materials and Methods

      Patients

      Two TMAs were used in this study. The first was a prostate cancer prognosis TMA containing prostatectomy specimens from 4699 consecutive patients undergoing radical prostatectomy between 1992 and 2008 at the Department of Urology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany (Table 1). This TMA is based on a previously described prostate cancer TMA consisting of 3261 samples,
      • El Gammal A.T.
      • Bruchmann M.
      • Zustin J.
      • Isbarn H.
      • Hellwinkel O.J.
      • Kollermann J.
      • Sauter G.
      • Simon R.
      • Wilczak W.
      • Schwarz J.
      • Bokemeyer C.
      • Brummendorf T.H.
      • Izbicki J.R.
      • Yekebas E.
      • Fisch M.
      • Huland H.
      • Graefen M.
      • Schlomm T.
      Chromosome 8p deletions and 8q gains are associated with tumor progression and poor prognosis in prostate cancer.
      with an additional 1438 tumors and updated clinical follow-up data. Clinical follow-up data were available for 4203 of the 4699 arrayed tumors. The median follow-up was 46.7 months (range, 1 to 219 months). None of the patients received neoadjuvant endocrine therapy. Salvage therapy was initiated in cases of biochemical relapse. In all patients, PSA values were measured quarterly in the first year, followed by biannual measurements in the second year and annual measurements after the third year following surgery. Recurrence was defined as a postoperative PSA of 0.2 ng/mL, increasing thereafter. The first PSA value of 0.2 ng/mL or greater was used to define the time of recurrence. Patients without evidence of tumor recurrence were censored at the last follow-up. The second TMA was constructed from 57 hormone-refractory prostate specimens collected from palliative transurethral resections at the Department of Urology, University Medical Center Hamburg-Eppendorf, and at the Department of Surgery, University of Montreal, Montreal, QC, Canada. Hormone-refractory prostate cancer was defined as follows: serum castration level of testosterone; three consecutive increases in the PSA level, resulting in two 50% increases over the nadir; anti-androgen withdrawal for at least 4 weeks; PSA progression despite secondary hormonal manipulation; or progression of osseous or soft tissue lesion. No follow-up data were available from these patients.
      Table 1Pathological and Clinical Data of Arrayed Prostate Cancers
      VariableStudy cohort receiving TMABiochemical relapse among categories
      (n = 4699)(n = 904)
      Follow-up (months)
       Mean56.6
       Median46.7
      Age (years)
       <5012622
       50–601399227
       61–702596520
       >7033784
      Pretreatment PSA (ng/mL)
       <466677
       4–102559376
       10–20894269
       >20289159
      pT category (AJCC 2002)
       pT23010272
       pT3a926286
       pT3b489307
       pT44239
      Gleason grade
       ≤3 + 31761125
       3 + 42055450
       4 + 3512261
       ≥4 + 413568
      pN category
       pN02317580
       pN+151111
      Surgical margin
       Negative3634573
       Positive810324
      Data are given as percentage or number of patients. Numbers do not always add up to 4699 in the different categories because of cases with missing data.
      AJCC, American Joint Committee on Cancer.

      PTEN FISH Analysis

      For PTEN deletion analysis, a dual-color FISH probe set was used. The set consisted of two SpectrumGreen-labeled bacterial artificial chromosome clones (RP11-380G5 and RP11-813O3; Source Bioscience, Nottingham, UK) and a SpectrumOrange-labeled commercial centromere 10 probe (06J36-090; Abbott, Wiesbaden, Germany) as a reference. Freshly cut TMA sections (4 μm thick) were deparaffinized and proteolytically pretreated using a commercial kit (paraffin pretreatment reagent kit; Abbott Molecular, Wiesbaden, Germany), followed by dehydration in 70%, 80%, and 96% ethanol, air drying, and denaturation for 10 minutes at 72°C in 70% formamide– two times standard saline citrate solution. Hybridization was performed overnight at 37°C in a humidified chamber; slides were then washed and counterstained with 0.2 μmol/L of DAPI in an antifade solution. Each tissue spot was evaluated, and the predominant signal was recorded for each FISH probe. A total of 659 tissue spots were excluded from FISH analysis because basal cell marker 34βE12 analysis
      • El Gammal A.T.
      • Bruchmann M.
      • Zustin J.
      • Isbarn H.
      • Hellwinkel O.J.
      • Kollermann J.
      • Sauter G.
      • Simon R.
      • Wilczak W.
      • Schwarz J.
      • Bokemeyer C.
      • Brummendorf T.H.
      • Izbicki J.R.
      • Yekebas E.
      • Fisch M.
      • Huland H.
      • Graefen M.
      • Schlomm T.
      Chromosome 8p deletions and 8q gains are associated with tumor progression and poor prognosis in prostate cancer.
      indicated lack of tumor cells. Thresholds for PTEN FISH analysis were established from 0.6-mm tissue spots from seven tumors with a known PTEN deletion (four with a heterozygous and three with a homozygous deletion), based on single-nucleotide polymorphism (SNP) array copy number analysis. In five of these tumors, PTEN signal losses by FISH were found in all analyzed tissue blocks. The two remaining cancers had tissue blocks with and without PTEN deletion, indicating the presence of intratumoral heterogeneity. In all seven cases, tumor blocks with PTEN deletion had FISH signal losses in most (at least 60%) tumor cells. According to these findings, homozygous deletion of PTEN was defined as complete absence of PTEN FISH probe signals in ≥60% of tumor nuclei of the tissue spot, with the presence of one or two PTEN FISH signals in adjacent normal cells. Tissue spots with a lack of PTEN signals in all (tumor and normal cells) or lack of any normal cells as an internal control for successful hybridization of the PTEN probe were excluded from analysis. Heterozygous deletion of PTEN was defined as the presence of fewer PTEN signals than centromere 10 probe signals of ≥60% tumor nuclei.

      PTEN IHC

      We tested seven different PTEN antibodies (Table 2) for their suitability in formalin-fixed, paraffin-embedded tissues. The antibody used for this study (ab31392, rabbit polyclonal; Abcam, Cambridge, UK) was selected because it showed reproducible nuclear staining. Freshly cut TMA sections were deparaffinized and incubated in pH 7.8 Tris-EDTA-citrate buffer at 121°C in an autoclave for antigen retrieval. The primary antibody was diluted 1:300 for IHC. Bound primary antibody was visualized using the DAKO EnVision Kit (Dako, Glostrup, Denmark). Nuclear staining intensity was estimated in a four-step scale: 0, negative; 1+, weak; 2+, moderate; and 3+, strong.
      Table 2Antibodies Tested for PTEN Expression Analysis
      CompanyCatalogue no.HostClonality
      DakoM3627MouseMonoclonal
      Abcamab32199RabbitMonoclonal
      AbnovaMAB7764MouseMonoclonal
      Cell Signaling Technology9559RabbitMonoclonal
      Abcamab31392RabbitPolyclonal
      Cell Signaling Technology9188RabbitMonoclonal
      AbnovaPAB12724RabbitPolyclonal

      ERG Analysis

      Immunohistochemistry (IHC) was used to detect ERG expression as a surrogate marker for ERG fusion. Freshly cut TMA sections were stained with a commercial anti-ERG antibody (clone EPR3864; dilution, 1:450; Epitomics, Burlingame, CA). Slides were deparaffinized and exposed to heat-induced antigen retrieval for 5 minutes in an autoclave at 121°C in pH 7.8 Tris-EDTA-citrate buffer. Bound primary antibody was visualized using the DAKO EnVision Kit. Tissue spots showing nuclear ERG staining in tumor cells were considered positive for ERG fusion. We have previously shown that ERG expression detected with this antibody shows 98.5% concordance with ERG rearrangement detected by FISH analysis.
      • Minner S.
      • Enodien M.
      • Sirma H.
      • Luebke A.M.
      • Krohn A.
      • Mayer P.S.
      • Simon R.
      • Tennstedt P.
      • Müller J.
      • Scholz L.
      • Brase J.C.
      • Liu A.Y.
      • Schlüter H.
      • Pantel K.
      • Schumacher U.
      • Bokemeyer C.
      • Steuber T.
      • Graefen M.
      • Sauter G.
      • Schlomm T.
      ERG status is unrelated to PSA recurrence in radically operated prostate cancer in the absence of antihormonal therapy.

      p53 and Ki-67 IHC Analysis

      Nuclear accumulation of p53 was analyzed by IHC, as previously described.
      • Schlomm T.
      • Iwers L.
      • Kirstein P.
      • Jessen B.
      • Kollermann J.
      • Minner S.
      • Passow-Drolet A.
      • Mirlacher M.
      • Milde-Langosch K.
      • Graefen M.
      • Haese A.
      • Steuber T.
      • Simon R.
      • Huland H.
      • Sauter G.
      • Erbersdobler A.
      Clinical significance of p53 alterations in surgically treated prostate cancers.
      The IHC data of Ki-67 were available from a previous study.
      • Minner S.
      • Jessen B.
      • Stiedenroth L.
      • Burandt E.
      • Kollermann J.
      • Mirlacher M.
      • Erbersdobler A.
      • Eichelberg C.
      • Fisch M.
      • Brummendorf T.H.
      • Bokemeyer C.
      • Simon R.
      • Steuber T.
      • Graefen M.
      • Huland H.
      • Sauter G.
      • Schlomm T.
      Low level HER2 overexpression is associated with rapid tumor cell proliferation and poor prognosis in prostate cancer.

      PTEN Mutational Analysis

      Tissue specimens were selected if at least 70% tumor cells were present. For DNA extraction, one core (0.6-mm diameter and 5-mm length) was taken from each tumor block. Paraffin was removed with xylene and 80% ethanol, followed by overnight digestion with proteinase K. DNA was isolated using a commercial kit (QIAamp DNA FFPE kit; Qiagen, Hilden, Germany). All nine PTEN exons were amplified by PCR using the AmpliTaq Gold polymerase (Applied Biosystems, Darmstadt, Germany). Primer sequences are given in Table 3. PCR cycling conditions included an initial denaturation step at 95°C for 10 minutes, followed by 35 cycles of 95°C denaturation for 20 seconds, 55°C or 53.5°C annealing for 20 seconds, 72°C extension for 40 seconds, and a final extension step at 72°C for 7 minutes. The quality of PCR products was verified by QIAxcel capillary electrophoresis (Qiagen). Sequencing was prepared by a Big Dye Terminator Kit (Applied Biosystems), and electrophoretic analysis was performed on the Genetic Analyzer 3100 (Applied Biosystems). Sequencing primers are given in Table 4.
      Table 3PTEN Exon-Specific PCR Primers
      ExonForward primerReverse primer
      15′-TTCCATCCTGCAGAAGAAGC-3′5′-CCCACGTTCTAAGAGAGTGACA-3′
      25′-CTCCAGCTATAGTGGGGAAAA-3′5′-CTTTTTCTGTGGCTTAGAAATC-3′
      35′-CCCATAGAAGGGGTATTTGTTG-3′5′-CTCTACCTCACTCTAACAAGCAGA-3′
      45′-CACATTATAAAGATTCAGGCAATGTT-3′5′-AAGATACAGTCTATCGGGTTTAAGTT-3′
      55′-CTTATTCTGAGGTTATCTTTTTACCAC-3′5′-TCCAGGAAGAGGAAAGGAAAA-3′
      65′-GGCTACGACCCAGTTACCAT-3′5′-GGAAGGATGAGAATTTCAAGCA-3′
      75′-CAGTTAAAGGCATTTCCTGTG-3′5′-TGGATATTTCTCCCAATGAAAG-3′
      8 fragment 15′-TGTTTAACATAGGTGACAGATTTTC-3′5′-AAGTCAACAACCCCCACAAA-3′
      8 fragment 25′-AGGTGACAGATTTTCTTTTTTA-3′5′-AAGTCAACAACCCCCACAAA-3′
      95′-GATGAGTCATATTTGTGGGTTTTC-3′5′-GGTCCATTTTCAGTTTATTCAAGT-3′
      Table 4PTEN Sequencing Primers
      ExonSequencing primer
      15′-TTCCATCCTGCAGAAGAAGC-3′
      25′-CTCCAGCTATAGTGGGGAAAA-3′
      35′-CCCATAGAAGGGGTATTTGTTG-3′
      45′-CACATTATAAAGATTCAGGCAATGTT-3′
      55′-TCCAGGAAGAGGAAAGGAAAA-3′
      65′-GGCTACGACCCAGTTACCAT-3′
      75′-CAGTTAAAGGCATTTCCTGTG-3′
      85′-AAGTCAACAACCCCCACAAA-3′
      95′-GGTCCATTTTCAGTTTATTCAAGT-3′

      SNP Array Analysis

      A total of 72 snap-frozen prostate cancer samples with at least 70% tumor cell content and five prostate cell lines (LNCaP, PC3, BPH, X22RV, and VCaP) were selected for SNP array analysis. DNA was isolated using a commercial kit (QIAamp DNA Mini Kit; Qiagen). Affymetrix SNP V6.0 arrays were used for copy number analysis. Fragmentation, labeling, and hybridization of the DNA to the SNP arrays were performed exactly as described in the Affymetrix V6.0 SNP array manual. We used our own genomic browser (FISH Oracle)
      • Mader M.
      • Simon R.
      • Steinbiss S.
      • Kurtz S.
      FISH Oracle: a web server for flexible visualization of DNA copy number data in a genomic context.
      to map all 10q23 deletions to the human genome reference sequence (Archive EnsEMBL release 54, May 2009) and to define the minimally overlapping region of deletion.

      Methylation Analysis

      Quantitative DNA methylation analysis at single CpG units was performed on 34 prostate cancers using MassARRAY EpiTyper (Sequenom, San Diego, CA), as previously described.
      • Ehrich M.
      • Nelson M.R.
      • Stanssens P.
      • Zabeau M.
      • Liloglou T.
      • Xinarianos G.
      • Cantor C.R.
      • Field J.K.
      • van den Boom D.
      Quantitative high-throughput analysis of DNA methylation patterns by base-specific cleavage and mass spectrometry.
      Briefly, bisulfite-treated genomic DNA was PCR amplified, in vitro transcribed, cleaved by RNase A, and subjected to matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (Sequenom, Hamburg, Germany). An overview of MassARRAY primer sequences for PCR amplicons covering the whole PTEN CpG island is given in Table 5. The location of primers used is shown in Supplemental Figure S1 (available at http://ajp.amjpathol.org). Detailed analyses were performed with two amplicons using the following primers: 5′-GATTTTTTTGGGGGTATYGGAG-3′ (forward) and 5′-CTCATCCRACTCCCTTACAAC-3′ (reverse) for PTEN4.5 and 5′-GTTGTTTATAGGYGTTGAGAGG-3′ (forward) and 5′-CCTCCCCTCRATCTTCC-3′ (reverse) for PTEN10p. GSTP1 methylation was analyzed as a control using the following primers: forward, 5′-GTGTGGTTTTTATTTYGGGTTTTTTTTT-3′; and reverse, 5′-TAACCCTAATACCAACAACATAC-3′. Y and R nucleotide codes denote wobble sites with C/T and A/G, respectively. Methylation standards (0%, 20%, 40%, 60%, 80%, and 100% methylated genomic DNA) and correction algorithms, based on custom scripts for the R statistical computing environment, were used for data normalization.
      Table 5Primers Used for MASSArray Analysis
      VariableForward primerReverse primer
      PTEN
       15′-TGGTATTAGTTTGGGGATTTTTT-3′5′-AAACTAATTACACAAACACCCA-3′
       25′-TGGGTGTTTGTGTAATTAGTTTTTTA-3′5′-AAACTACTTTCCAAAAAAAATCAC-3′
       35′-GGAAAGTAGTTTYGATTGTGGTT-3′5′-ATTCTCAAAAACCACCTAACCCC-3′
       45′-GGGGTTAGGTGGTTTTTGAGAAT-3′5′-CAAAAACCCAAAAAACACCTATCTA-3′
       4.55′-GATTTTTTTGGGGGTATYGGAG-3′5′-CTCATCCRACTCCCTTACAAC-3′
       55′-TAGATAGGTGTTTTTTGGGTTTTTG-3′5′-TTCCCCCAAATCTATATCCTCATAAT-3′
       65′-ATTATGAGGATATAGATTTGGGGGAA-3′5′-CCCTACAAAAAAAATACCCTCC-3′
       75′-GGAGGGTATTTTTTTTGTAGGG-3′5′-ACCTCTACCCAAAAACCCAA-3′
       85′-TTGGGTTTTTGGGTAGAGGT-3′5′-CATACCCAATATAACTACCTAAAACTTACT-3′
       9p5′-GGAAGATYGAGGGGAGG-3′5′-CAAACCCCAAACAACTACAC-3′
       10p5′-GTTGTTTATAGGYGTTGAGAGG-3′5′-CCTCCCCTCRATCTTCC-3′
       11p5′-GAGAAGTYGAGGAAGAGGT-3′5′-CCTCTCAACRCCTATAAACAAC-3′
       12p5′-TTTTTTGAAAGGGAAGGTGGAA-3′5′-TCCCAACCCTAAAAATAATAACAAA-3′
       13p5′-TTTGTTATTATTTTTAGGGTTGGGA-3′5′-AAATAAAAAAAAAACRAATAATCCTCC-3′
       14p5′-GGAGGATTATTYGTTTTTTTTTTATTT-3′5′-CTACTAATAACRAAACTTCTTCTAC-3′
      GSTP1
       GSTP1_6ss5′-GTGTGGTTTTTATTTYGGGTTTTTTTTT-3′5′-TAACCCTAATCTACCAACAACATAC-3′
      Y and R nucleotide codes stand for wobble sites with C/T and A/G, respectively.

      Statistics

      For statistical analysis, the JMP 8.0 software (SAS Institute Inc., Cary, NC) was used. Contingency tables were calculated to study the association between PTEN deletion and clinicopathological variables, and the χ2 (likelihood) test was used to find significant relationships. Kaplan-Meier curves were generated for PSA recurrence-free survival. The log-rank test was applied to test the significance of differences between stratified survival functions. Cox proportional hazards regression analysis was performed to test the statistical independence and significance between pathological, molecular, and clinical variables.

      Results

      Technical Issues

      A total of 4040 hormone-naïve and 57 hormone-refractory cancers were included in FISH analysis in this study. Analysis failed in 45% hormone-naïve and in 14% hormone-refractory tumors, because of either lack of tissue spots in the TMA section or faint or lacking FISH signals. In summary, 2217 hormone-naïve and 49 hormone-refractory tumors were successfully analyzed and included in the statistical analyses.

      Prevalence and Type of PTEN Deletions and Association to Prostate Cancer Phenotype

      PTEN deletions were found in 20.2% (458/2266) of all prostate cancers (Figure 1, A–C). Overall, homozygous PTEN deletions (12.1%) were slightly more frequent than heterozygous PTEN deletions (8.1%). Both heterozygous and homozygous deletions were more frequent in hormone-refractory compared with hormone-naïve cancers (P < 0.0001 for each). The difference was particularly strong for homozygous deletions, which were found in 16 (32.7%) of 49 hormone-refractory cancers, but only in 259 (11.7%) of 2217 hormone-naïve tumors (P < 0.0001). The relationship between PTEN deletions and tumor phenotype and clinical parameters is summarized in Table 6. PTEN deletions (including heterozygous and homozygous deletions) were significantly linked to advanced tumor stage (P < 0.0001), high Gleason grade (P < 0.0001), presence of lymph node metastasis (P = 0.0002), and positive surgical margin (P = 0.0462). Although a significant P value was obtained for the association between PTEN deletions and PSA serum level (P = 0.0043), we did not consider this result as indicative for a true relationship because the different PSA levels were not unequivocally paralleled by an increase or decrease of PTEN deletions.
      Figure thumbnail gr1
      Figure 1Examples for PTEN deletions in prostate cancer. A: Normal copy number of PTEN with two green PTEN and 2 red centromer 10 signals. B: PTEN heterozygous deletion with one green PTEN signal and two red centromer 10 signals. C: PTEN homozygous deletion completely lacking PTEN signals but showing two red centromer 10 signals. Arrow, normal prostate cells showing normal PTEN copy number.
      Table 6Clinicopathological Associations of PTEN Deletions
      ParameternEvaluablePTEN deletion status (%)P value
      HeterozygousHomozygous
      All cancers475622668.112.1
      Tumor stage
       pT2301013425.17.9<0.0001
       pT3a92649110.616.3
       pT3b48927816.921.2
       pT442205.035.0
       HR574912.232.7<0.0001
      HR versus hormone-naïve prostate cancer.
      Gleason grade
      HR cancers excluded.
       ≤3 + 317617154.55<0.0001
       3 + 4205510398.613
       4 + 351227811.921.6
       ≥4 + 41359714.420.6
      Lymph node metastasis
      HR cancers excluded.
       N0231711119.613.40.0002
       N+1519912.129.3
      Preoperative PSA level (ng/mL)
      HR cancers excluded.
       <466624910.513.20.0043
       4–1025599045.911.5
       10–208943739.810.6
       >2028914312.810.1
      Surgical margin
      HR cancers excluded.
       Negative363413417.411.10.0462
       Positive8103539.414.4
      HR, hormone refractory.
      low asterisk HR versus hormone-naïve prostate cancer.
      HR cancers excluded.

      Association of PTEN Deletions to Other Molecular Markers of Prostate Cancer

      Data on PTEN and p53 status were available from 1798 hormone-naïve cancers. PTEN deletions were significantly more frequent in p53-positive tumors (heterozygous, 10.5%; and homozygous, 36.8%) than in p53-negative tumors (heterozygous, 8.4%; and homozygous, 11.2%; P < 0.0001; Figure 2A). This overall significant association resulted from homozygous deletions (P < 0.0001), whereas the difference was not significant for heterozygous deletions (P = 0.2758). Data on PTEN deletion and ERG fusion status were available from 2177 tumors. PTEN deletion was strongly associated with ERG fusion-positive tumors (29.1% versus 10.7%; P < 0.0001 overall) and for separate analysis of heterozygous and homozygous deletions (Figure 2B). PTEN and Ki-67 labeling index (Ki-67 LI) data were both available from 1802 tumors. Ki-67 LI was significantly higher in PTEN- deleted (average Ki-67 LI, 6.3) than in -undeleted (average Ki-67 LI, 5.5; P = 0.0321) cancers, if all cancers were jointly analyzed, but there was no statistically significant association found in tumors of identical stage and grade (see Supplemental Table S1 at http://ajp.amjpathol.org).
      Figure thumbnail gr2
      Figure 2Association of PTEN deletion status to p53 expression (P < 0.0001) (A) and ERG fusion status (P < 0.0001) (B). Normal includes two PTEN gene copies. Heterozygous, heterozygous deletion with one PTEN gene copy; homozygous, homozygous deletion completely lacking PTEN.

      Clinical Significance of PTEN Deletions, ERG Fusion, and p53

      PTEN FISH was analyzable in a subset of 1931 cases with follow-up data. In this subset, Gleason grade, pT stage, and preoperative serum PSA levels were significantly linked to poor prognosis (P < 0.0001, data not shown). PTEN deletions were significantly linked to early PSA recurrence in univariate analysis (P < 0.0001, Figure 3A). No difference was seen between tumors with heterozygous or homozygous deletion (P = 0.6970). In a multivariate cyclooxygenase regression proportional hazard analysis including pT stage, Gleason grade, preoperative PSA level, and PTEN deletion status, PTEN deletion was identified as an independent predictor of PSA recurrence-free survival (P = 0.0158, Table 7). ERG fusion was analyzable in 3751 tumors with follow-up data. The presence of ERG fusion was unrelated to patient prognosis (P = 0.7346, Figure 3B). A combined analysis of PTEN and ERG in 1895 tumors revealed no prognostic differences between tumors with PTEN deletion and ERG fusion, compared with tumors with PTEN deletion but lack of ERG fusion (P = 0.9459, Figure 3C). There was a significantly worse prognosis for ERG-negative compared with ERG-positive tumors in the subset of 1524 cancers with normal PTEN copy numbers (P = 0.0044). A combined analysis of PTEN deletion and presence of nuclear accumulation of p53 in a subset of 1545 cancers revealed that 35 cancers with p53 accumulation (irrespective of the PTEN deletion status) had a significantly worse prognosis than tumors with PTEN deletion but lack of p53 alteration (Figure 3D, P = 0.0162).
      Figure thumbnail gr3
      Figure 3Association between PTEN deletion (A), ERG fusion (B), and the combination of PTEN deletion and ERG fusion (C) or nuclear p53 accumulation with biochemical recurrence in prostate cancer (D).
      Table 7COX Regression Multivariate Analysis for Predictive Factor Biochemical Recurrence
      ParameterFactorHR95% CIP value
      Gleason grade3 + 4 versus ≤3 + 32.31.71–3.76<0.0001
      4 + 3 versus ≤3 + 35.74.01–8.10
      ≥4 + 4 versus ≤3 + 36.13.76–9.69
      pT stagepT3a versus pT21.91.47–2.43<0.0001
      pT3b versus pT23.22.42–4.14
      pT4 versus pT25.93.37–9.83
      PSA level4–9 versus <40.90.64–1.29<0.0001
      10–20 versus <41.41.00–2.05
      >20 versus <41.81.18–2.66
      PTENDeleted versus not deleted1.31.05–1.600.0158
      HR, hazard ratio.

      PTEN IHC

      PTEN IHC was successful in 3320 of the 4699 arrayed hormone-naïve primary prostate cancers. Immunostaining was considered negative in 83 cases (2.5%), weak in 903 cases (27.2%), moderate in 2192 cases (66.0%), and strong in 142 cases (4.3%) (see Supplemental Table S2 at http://ajp.amjpathol.org). No meaningful associations were found between the PTEN staining levels and tumor phenotype or presence of PTEN deletions (see Supplemental Table S1 and Supplemental Figures S2 and S3, A and B, at http://ajp.amjpathol.org). The intensity of cytoplasmic staining was markedly reduced with higher (1:150) antibody dilutions (see Supplemental Figure S3 at http://ajp.amjpathol.org). PTEN staining was also unrelated to patient prognosis in a Kaplan-Meier survival analysis (P = 0.5251, data not shown).

      Mutation Analysis of PTEN

      All nine exons of PTEN were analyzed in 97 prostate cancers containing 71 hormone-naïve and 26 hormone-refractory prostate cancers. PTEN mutations were found in 7 cases, including 5 (7.0%) hormone-naïve and 2 (7.7%) hormone-refractory tumors (P = 0.943). Mutations were found in tumors with heterozygous PTEN deletion in 12.9% (4/31, one hormone refractory) and in tumors with normal PTEN copy numbers in 2% (1/59, one hormone refractory, P = 0.027, Table 8). No FISH result was available for two additional cases with mutation. The five hormone-naïve mutated tumors had a Gleason score of 6 to 7. Based on the type of mutation identified in our analysis, at least four of the seven mutations inevitably cause PTEN inactivation. These include three tumors with small deletions and insertions in exons 1 and 8, causing frameshift mutations, and another tumor with a truncating mutation (E201end) in exon 8. The remaining three tumors showed point mutations in exons 3, 5, and 8, including amino acid changes from tyrosine to asparaginic acid (T68G) in exon 3; from asparaginic acid to asparagine (D326N) in exon 8; and from histidine to tyrosine (H118Y) in exon 5 (see Supplemental Figure S4, A–E, at http://ajp.amjpathol.org). Most likely, the latter two mutations also led to inactivation, because exon 5 contained the functionally relevant WDP and P loops that formed the active pocket of the phosphatase domain.
      Table 8Association between PTEN Mutations and PTEN Deletions
      VariablenPTEN not mutated
      Data are given as number (percentage).
      PTEN mutated
      Data are given as number (percentage).
      P value
      PTEN not deleted5958 (98)1 (2)0.027
      PTEN heterozygous deleted versus not deleted.
      PTEN heterozygous deleted3127 (87)4 (13)
      Deletion status unknown75 (71)2 (29)
      Total9790 (93)7 (7)
      low asterisk Data are given as number (percentage).
      PTEN heterozygous deleted versus not deleted.

      Architecture of 10q23 Deletions Harboring PTEN

      Deletions involving the long arm of chromosome 10 were found in 14 of the 77 analyzed prostate cancer samples. The largest deletion spanned >23 Mb. The smallest deletion marked a region of 809 kb that were commonly deleted in all 14 tumors. This region contained the PTEN gene and two adjacent genes (ATAD1 and RNLS). The minimal overlapping region of deletion also contained both bacterial artificial chromosome clones used for generation of our FISH probe (Figure 4).
      Figure thumbnail gr4
      Figure 4Genomic PTEN deletions detected in 14 of 77 prostate cancer samples in SNP-array analysis. Left panel: An overview of position and size of genomic deletion relative to the 10q region in 14 prostate cancers. Right panel: Detailed view of genomic deleted region relative to 10q23.2 to 10q23.31 region. The position of bacterial artificial chromosome clones (RP11-380G5 and RP11-813O3) relative to the PTEN locus is indicated. ≪ and ≫, the deletion exceeds the genomic area depicted in the figure.

      Epigenetic Changes at the PTEN Promoter

      We designed 15 primer pairs to cover the whole CpG island associated with PTEN for quantitative DNA methylation analyses using MassARRAY technology. Initial analyses indicated overall low methylation and did not reveal any differences between tumor and normal samples (data not shown). We selected two amplicons (PTEN4.5 and PTEN10p) covering 6 and 7 CpG units, each representing one or two individual CpG sites, to further analyze PTEN promoter methylation in 34 prostate cancer samples and 5 normal prostate tissues (Figure 5A). Median methylation was <10% in both amplicons and did not differ between tumor and normal samples (Figure 5B). In contrast, all tumor samples were highly methylated at the GSTP1 promoter CpG island analyzed as a positive control, with median methylation of 79% in tumor samples and 5% in normal controls.
      Figure thumbnail gr5
      Figure 5PTEN promoter methylation. A: Representative heat map of quantitative CpG methylation analysis using MassARRAY technology. Each row represents a sample of normal prostate tissue (N) or prostate cancer (P). Each square represents a single CpG site or a group of one to two CpG sites analyzed together. Methylation frequencies extend from light blue (0%) to dark blue (100%) and are corrected, compared with a methylation standard with 0% to 100% methylation. GSTP1 is analyzed as a methylation control. B: Dot blot of average methylation across amplicons. Horizontal lines, median methylation in tumor and normal samples, respectively.

      Discussion

      Our study shows that PTEN deletions occur more frequently (20%) than mutations (8%) in prostate cancer. PTEN deletions are strongly linked to important biological and clinical features, such as rapid tumor progression, hormone-refractory state, and early PSA recurrence. The low rate of mutations fits well with previous reports showing that 2.5%,
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      • Horvath S.
      • Smith B.L.
      • Crosby K.
      • Lebel L.A.
      • Schrage M.
      • Said J.
      • De Kernion J.
      • Reiter R.E.
      • Sawyers C.L.
      Antibody-based profiling of the phosphoinositide 3-kinase pathway in clinical prostate cancer.
      • Bedolla R.
      • Prihoda T.J.
      • Kreisberg J.I.
      • Malik S.N.
      • Krishnegowda N.K.
      • Troyer D.A.
      • Ghosh P.M.
      Determining risk of biochemical recurrence in prostate cancer by immunohistochemical detection of PTEN expression and Akt activation.
      • McCall P.
      • Witton C.J.
      • Grimsley S.
      • Nielsen K.V.
      • Edwards J.
      Is PTEN loss associated with clinical outcome measures in human prostate cancer?.
      • Koumakpayi I.H.
      • Le Page C.
      • Mes-Masson A.M.
      • Saad F.
      Hierarchical clustering of immunohistochemical analysis of the activated ErbB/PI3K/Akt/NF-kappaB signalling pathway and prognostic significance in prostate cancer.
      • Osman I.
      • Dai J.
      • Mikhail M.
      • Navarro D.
      • Taneja S.S.
      • Lee P.
      • Christos P.
      • Shen R.
      • Nanus D.M.
      Loss of neutral endopeptidase and activation of protein kinase B (Akt) is associated with prostate cancer progression.
      Accordingly, associations between the level of PTEN expression and clinicopathological parameters vary. For example, McCall et al
      • McCall P.
      • Witton C.J.
      • Grimsley S.
      • Nielsen K.V.
      • Edwards J.
      Is PTEN loss associated with clinical outcome measures in human prostate cancer?.
      reported a link between low-level cytoplasmic PTEN expression and tumor recurrence, and between loss of nuclear PTEN staining and patient survival in 68 hormone-naïve prostate cancers. However, Koumakpayi et al
      • Koumakpayi I.H.
      • Le Page C.
      • Mes-Masson A.M.
      • Saad F.
      Hierarchical clustering of immunohistochemical analysis of the activated ErbB/PI3K/Akt/NF-kappaB signalling pathway and prognostic significance in prostate cancer.
      and Bedolla et al
      • Bedolla R.
      • Prihoda T.J.
      • Kreisberg J.I.
      • Malik S.N.
      • Krishnegowda N.K.
      • Troyer D.A.
      • Ghosh P.M.
      Determining risk of biochemical recurrence in prostate cancer by immunohistochemical detection of PTEN expression and Akt activation.
      could not confirm such associations in similarly sized patient cohorts. A link between loss of PTEN expression and high Gleason grade or advanced tumor stage was reported from some studies,
      • McMenamin M.E.
      • Soung P.
      • Perera S.
      • Kaplan I.
      • Loda M.
      • Sellers W.R.
      Loss of PTEN expression in paraffin-embedded primary prostate cancer correlates with high Gleason score and advanced stage.
      • Bertram J.
      • Peacock J.W.
      • Fazli L.
      • Mui A.L.
      • Chung S.W.
      • Cox M.E.
      • Monia B.
      • Gleave M.E.
      • Ong C.J.
      Loss of PTEN is associated with progression to androgen independence.
      but not corroborated by other studies.
      • Thomas G.V.
      • Horvath S.
      • Smith B.L.
      • Crosby K.
      • Lebel L.A.
      • Schrage M.
      • Said J.
      • De Kernion J.
      • Reiter R.E.
      • Sawyers C.L.
      Antibody-based profiling of the phosphoinositide 3-kinase pathway in clinical prostate cancer.
      • Bedolla R.
      • Prihoda T.J.
      • Kreisberg J.I.
      • Malik S.N.
      • Krishnegowda N.K.
      • Troyer D.A.
      • Ghosh P.M.
      Determining risk of biochemical recurrence in prostate cancer by immunohistochemical detection of PTEN expression and Akt activation.
      • Osman I.
      • Dai J.
      • Mikhail M.
      • Navarro D.
      • Taneja S.S.
      • Lee P.
      • Christos P.
      • Shen R.
      • Nanus D.M.
      Loss of neutral endopeptidase and activation of protein kinase B (Akt) is associated with prostate cancer progression.
      PTEN deletions were approximately three times more frequent in ERG-positive compared with ERG-negative cancers. This association was not because of a higher fraction of advanced tumors in the subset of ERG fusion-positive cancers, because stage and grade distribution was comparable in both subsets (see Supplemental Table S3 at http://ajp.amjpathol.org). This is in line with recent studies
      • King J.C.
      • Xu J.
      • Wongvipat J.
      • Hieronymus H.
      • Carver B.S.
      • Leung D.H.
      • Taylor B.S.
      • Sander C.
      • Cardiff R.D.
      • Couto S.S.
      • Gerald W.L.
      • Sawyers C.L.
      Cooperativity of TMPRSS2-ERG with PI3-kinase pathway activation in prostate oncogenesis.
      • Carver B.S.
      • Tran J.
      • Gopalan A.
      • Chen Z.
      • Shaikh S.
      • Carracedo A.
      • Alimonti A.
      • Nardella C.
      • Varmeh S.
      • Scardino P.T.
      • Cordon-Cardo C.
      • Gerald W.
      • Pandolfi P.P.
      Aberrant ERG expression cooperates with loss of PTEN to promote cancer progression in the prostate.
      • Han B.
      • Mehra R.
      • Lonigro R.J.
      • Wang L.
      • Suleman K.
      • Menon A.
      • Palanisamy N.
      • Tomlins S.A.
      • Chinnaiyan A.M.
      • Shah R.B.
      Fluorescence in situ hybridization study shows association of PTEN deletion with ERG rearrangement during prostate cancer progression.
      • Yoshimoto M.
      • Joshua A.M.
      • Cunha I.W.
      • Coudry R.A.
      • Fonseca F.P.
      • Ludkovski O.
      • Zielenska M.
      • Soares F.A.
      • Squire J.A.
      Absence of TMPRSS2: ERG fusions and PTEN losses in prostate cancer is associated with a favorable outcome.
      reporting a link between PTEN deletion and ERG fusion in prostate cancer. Our findings suggest a selection advantage for tumor cells harboring both PTEN deletion and ERG fusion. Several studies using transgenic mice to monitor the effects of PTEN inactivation and/or ERG expression suggested a cooperative effect of these genes for prostate cancer initiation and progression. For example, Kwabi-Addo et al
      • Kwabi-Addo B.
      • Giri D.
      • Schmidt K.
      • Podsypanina K.
      • Parsons R.
      • Greenberg N.
      • Ittmann M.
      Haploinsufficiency of the Pten tumor suppressor gene promotes prostate cancer progression.
      found that PTEN levels corresponding to heterozygous PTEN deletion caused PIN, and Trotman et al
      • Trotman L.C.
      • Niki M.
      • Dotan Z.A.
      • Koutcher J.A.
      • Di Cristofano A.
      • Xiao A.
      • Khoo A.S.
      • Roy-Burman P.
      • Greenberg N.M.
      • Van Dyke T.
      • Cordon-Cardo C.
      • Pandolfi P.P.
      Pten dose dictates cancer progression in the prostate.
      reported that particularly low PTEN levels (ie, 25% of wild-type expression) were sufficient for development of invasive cancer. Similarly, overexpression of ERG
      • Tomlins S.A.
      • Laxman B.
      • Varambally S.
      • Cao X.
      • Yu J.
      • Helgeson B.E.
      • Cao Q.
      • Prensner J.R.
      • Rubin M.A.
      • Shah R.B.
      • Mehra R.
      • Chinnaiyan A.M.
      Role of the TMPRSS2-ERG gene fusion in prostate cancer.
      • Zong Y.
      • Xin L.
      • Goldstein A.S.
      • Lawson D.A.
      • Teitell M.A.
      • Witte O.N.
      ETS family transcription factors collaborate with alternative signaling pathways to induce carcinoma from adult murine prostate cells.
      or ETV1
      • Tomlins S.A.
      • Laxman B.
      • Dhanasekaran S.M.
      • Helgeson B.E.
      • Cao X.
      • Morris D.S.
      • Menon A.
      • Jing X.
      • Cao Q.
      • Han B.
      • Yu J.
      • Wang L.
      • Montie J.E.
      • Rubin M.A.
      • Pienta K.J.
      • Roulston D.
      • Shah R.B.
      • Varambally S.
      • Mehra R.
      • Chinnaiyan A.M.
      Distinct classes of chromosomal rearrangements create oncogenic ETS gene fusions in prostate cancer.
      alone resulted in PIN in some studies, although King et al
      • King J.C.
      • Xu J.
      • Wongvipat J.
      • Hieronymus H.
      • Carver B.S.
      • Leung D.H.
      • Taylor B.S.
      • Sander C.
      • Cardiff R.D.
      • Couto S.S.
      • Gerald W.L.
      • Sawyers C.L.
      Cooperativity of TMPRSS2-ERG with PI3-kinase pathway activation in prostate oncogenesis.
      observed PIN only if ERG was overexpressed in mice that were either PTEN deficient or had high AKT activity. ERG overexpression in PTEN-deficient mice of Carver et al
      • Carver B.S.
      • Tran J.
      • Gopalan A.
      • Chen Z.
      • Shaikh S.
      • Carracedo A.
      • Alimonti A.
      • Nardella C.
      • Varmeh S.
      • Scardino P.T.
      • Cordon-Cardo C.
      • Gerald W.
      • Pandolfi P.P.
      Aberrant ERG expression cooperates with loss of PTEN to promote cancer progression in the prostate.
      caused PIN and led to invasive cancer. Although the link between PTEN deletion and ERG fusion found in our study supports the existence of such cooperative effects, it also suggests that ERG fusion is not required for PTEN loss to determine aggressive tumor behavior, because PTEN deletion in both ERG fusion-positive and fusion-negative cancers was independently linked to poor prognosis. Two previous studies
      • Yoshimoto M.
      • Joshua A.M.
      • Cunha I.W.
      • Coudry R.A.
      • Fonseca F.P.
      • Ludkovski O.
      • Zielenska M.
      • Soares F.A.
      • Squire J.A.
      Absence of TMPRSS2: ERG fusions and PTEN losses in prostate cancer is associated with a favorable outcome.
      • Reid A.H.
      • Attard G.
      • Ambroisine L.
      • Fisher G.
      • Kovacs G.
      • Brewer D.
      • Clark J.
      • Flohr P.
      • Edwards S.
      • Berney D.M.
      • Foster C.S.
      • Fletcher A.
      • Gerald W.L.
      • Moller H.
      • Reuter V.E.
      • Scardino P.T.
      • Cuzick J.
      • de Bono J.S.
      • Cooper C.S.
      Molecular characterisation of ERG, ETV1 and PTEN gene loci identifies patients at low and high risk of death from prostate cancer.
      have analyzed the association between co-alterations of PTEN and ERG and patient prognosis. The results differed in that Yoshimoto et al
      • Yoshimoto M.
      • Joshua A.M.
      • Cunha I.W.
      • Coudry R.A.
      • Fonseca F.P.
      • Ludkovski O.
      • Zielenska M.
      • Soares F.A.
      • Squire J.A.
      Absence of TMPRSS2: ERG fusions and PTEN losses in prostate cancer is associated with a favorable outcome.
      found the worst prognosis for patients with both PTEN deletion and ERG fusion in a study on 125 patients, whereas Reid et al
      • Reid A.H.
      • Attard G.
      • Ambroisine L.
      • Fisher G.
      • Kovacs G.
      • Brewer D.
      • Clark J.
      • Flohr P.
      • Edwards S.
      • Berney D.M.
      • Foster C.S.
      • Fletcher A.
      • Gerald W.L.
      • Moller H.
      • Reuter V.E.
      • Scardino P.T.
      • Cuzick J.
      • de Bono J.S.
      • Cooper C.S.
      Molecular characterisation of ERG, ETV1 and PTEN gene loci identifies patients at low and high risk of death from prostate cancer.
      suggested, in their analysis of 308 patients, that tumors with PTEN loss but lack of ERG fusion had a particularly poor outcome. Our study demonstrates that PTEN is a major strong driver of patient prognosis, independent of ERG status. Given the strong association seen between PTEN deletions and high Ki-67 labeling index, the aggressive behavior of PTEN-deleted cancers may be driven by increased cell proliferation. Such a scenario would also be concordant with the known role of PTEN as a key regulator of the AKT growth-signaling pathway.
      • Stambolic V.
      • Suzuki A.
      • de la Pompa J.L.
      • Brothers G.M.
      • Mirtsos C.
      • Sasaki T.
      • Ruland J.
      • Penninger J.M.
      • Siderovski D.P.
      • Mak T.W.
      Negative regulation of PKB/Akt-dependent cell survival by the tumor suppressor PTEN.
      Remarkably, ERG-negative cancers had earlier PSA recurrence than ERG-positive cancers if tumors were PTEN wild type, whereas the ERG status had no detectable impact on clinical outcome if all tumors were analyzed together. This observation may be related to both the higher fraction of PTEN-deleted tumors in ERG-positive compared with ERG-negative cancers and to the presence of other molecular alterations that may drive poor prognosis, particularly in ERG-negative cancers.
      Nuclear accumulation of p53 is strongly linked to presence of inactivating p53 mutations,
      • Schlomm T.
      • Iwers L.
      • Kirstein P.
      • Jessen B.
      • Kollermann J.
      • Minner S.
      • Passow-Drolet A.
      • Mirlacher M.
      • Milde-Langosch K.
      • Graefen M.
      • Haese A.
      • Steuber T.
      • Simon R.
      • Huland H.
      • Sauter G.
      • Erbersdobler A.
      Clinical significance of p53 alterations in surgically treated prostate cancers.
      which is an important reason for failure of cellular repair systems and development of genetic instability.
      • Song H.
      • Xu Y.
      Gain of function of p53 cancer mutants in disrupting critical DNA damage response pathways.
      The association between PTEN deletions and nuclear p53 accumulation suggests that development of PTEN deletion may be caused by p53-mediated genetic instability in a subset of prostate cancers. The particular striking association between homozygous PTEN deletion and nuclear p53 accumulation further suggests a selection advantage for complete PTEN inactivation in a p53-deficient background. This finding fits well with a previous report
      • Chen Z.
      • Trotman L.C.
      • Shaffer D.
      • Lin H.K.
      • Dotan Z.A.
      • Niki M.
      • Koutcher J.A.
      • Scher H.I.
      • Ludwig T.
      • Gerald W.
      • Cordon-Cardo C.
      • Pandolfi P.P.
      Crucial role of p53-dependent cellular senescence in suppression of Pten-deficient tumorigenesis.
      describing a functional link between complete PTEN inactivation and subsequent activation of a p53-dependent failsafe program, which triggers a proliferation block and induces cellular senescence. In their prostate cancer mouse model, Chen et al
      • Chen Z.
      • Trotman L.C.
      • Shaffer D.
      • Lin H.K.
      • Dotan Z.A.
      • Niki M.
      • Koutcher J.A.
      • Scher H.I.
      • Ludwig T.
      • Gerald W.
      • Cordon-Cardo C.
      • Pandolfi P.P.
      Crucial role of p53-dependent cellular senescence in suppression of Pten-deficient tumorigenesis.
      found that invasive tumors developed only in mice with concurrent inactivation of both p53 and PTEN. The comparison of impacts of PTEN and p53 on patient outcome emphasizes the striking prognostic relevance of p53 alterations on prostate cancer outcome, as previously described by us
      • Schlomm T.
      • Iwers L.
      • Kirstein P.
      • Jessen B.
      • Kollermann J.
      • Minner S.
      • Passow-Drolet A.
      • Mirlacher M.
      • Milde-Langosch K.
      • Graefen M.
      • Haese A.
      • Steuber T.
      • Simon R.
      • Huland H.
      • Sauter G.
      • Erbersdobler A.
      Clinical significance of p53 alterations in surgically treated prostate cancers.
      and other groups.
      • Zellweger T.
      • Ninck C.
      • Bloch M.
      • Mirlacher M.
      • Koivisto P.A.
      • Helin H.J.
      • Mihatsch M.J.
      • Gasser T.C.
      • Bubendorf L.
      Expression patterns of potential therapeutic targets in prostate cancer.
      • Visakorpi T.
      • Kallioniemi O.P.
      • Heikkinen A.
      • Koivula T.
      • Isola J.
      Small subgroup of aggressive, highly proliferative prostatic carcinomas defined by p53 accumulation.
      • Henke R.P.
      • Kruger E.
      • Ayhan N.
      • Hubner D.
      • Hammerer P.
      • Huland H.
      Immunohistochemical detection of p53 protein in human prostatic cancer.
      Our data do not suggest an additional prognostic impact of PTEN deletions in p53-altered cancers, whereas PTEN deletions remain of high prognostic importance in p53-negative cancers.
      Data from our SNP array copy number analysis demonstrate that the minimal commonly deleted region at 10q23 in prostate cancer contains only one gene, PTEN. We took advantage of the fact that the minimal region of deletion always extended the PTEN gene locus and constructed a large (360-kb) FISH probe, including flanking regions of PTEN, to obtain bright FISH signals that can be scored with high reliability. By using this probe, the fraction of PTEN deletions detected in our study in hormone-naïve (18%) and in hormone-refractory (45%) cancers is in the lower range of previous studies
      • Han B.
      • Mehra R.
      • Lonigro R.J.
      • Wang L.
      • Suleman K.
      • Menon A.
      • Palanisamy N.
      • Tomlins S.A.
      • Chinnaiyan A.M.
      • Shah R.B.
      Fluorescence in situ hybridization study shows association of PTEN deletion with ERG rearrangement during prostate cancer progression.
      • Yoshimoto M.
      • Cutz J.C.
      • Nuin P.A.
      • Joshua A.M.
      • Bayani J.
      • Evans A.J.
      • Zielenska M.
      • Squire J.A.
      Interphase FISH analysis of PTEN in histologic sections shows genomic deletions in 68% of primary prostate cancer and 23% of high-grade prostatic intra-epithelial neoplasias.
      • Yoshimoto M.
      • Joshua A.M.
      • Cunha I.W.
      • Coudry R.A.
      • Fonseca F.P.
      • Ludkovski O.
      • Zielenska M.
      • Soares F.A.
      • Squire J.A.
      Absence of TMPRSS2: ERG fusions and PTEN losses in prostate cancer is associated with a favorable outcome.
      • McCall P.
      • Witton C.J.
      • Grimsley S.
      • Nielsen K.V.
      • Edwards J.
      Is PTEN loss associated with clinical outcome measures in human prostate cancer?.
      • Sircar K.
      • Yoshimoto M.
      • Monzon F.A.
      • Koumakpayi I.H.
      • Katz R.L.
      • Khanna A.
      • Alvarez K.
      • Chen G.
      • Darnel A.D.
      • Aprikian A.G.
      • Saad F.
      • Bismar T.A.
      • Squire J.A.
      PTEN genomic deletion is associated with p-Akt and AR signalling in poorer outcome, hormone refractory prostate cancer.
      • Verhagen P.C.
      • van Duijn P.W.
      • Hermans K.G.
      • Looijenga L.H.
      • van Gurp R.J.
      • Stoop H.
      • van der Kwast T.H.
      • Trapman J.
      The PTEN gene in locally progressive prostate cancer is preferentially inactivated by bi-allelic gene deletion.
      • Yoshimoto M.
      • Cunha I.W.
      • Coudry R.A.
      • Fonseca F.P.
      • Torres C.H.
      • Soares F.A.
      • Squire J.A.
      FISH analysis of 107 prostate cancers shows that PTEN genomic deletion is associated with poor clinical outcome.
      • Reid A.H.
      • Attard G.
      • Ambroisine L.
      • Fisher G.
      • Kovacs G.
      • Brewer D.
      • Clark J.
      • Flohr P.
      • Edwards S.
      • Berney D.M.
      • Foster C.S.
      • Fletcher A.
      • Gerald W.L.
      • Moller H.
      • Reuter V.E.
      • Scardino P.T.
      • Cuzick J.
      • de Bono J.S.
      • Cooper C.S.
      Molecular characterisation of ERG, ETV1 and PTEN gene loci identifies patients at low and high risk of death from prostate cancer.
      reporting 17% to 68% PTEN deletion in localized and 41% to 77% in PTEN deletion in hormone-refractory cancers. We believe that the comparatively low frequency of deletions in our study is mainly caused by stringent criteria for defining PTEN deletions. We expected FISH signal loss in at least 60% of tumor cells to call a tumor deleted. This threshold was based on FISH findings in seven tumors with known heterozygous or homozygous deletions, according to an SNP array-based copy number analysis. In 0.6-mm tissue spots obtained from these tumors, virtually all tumor cells showed PTEN signal losses, including two cancers that had both tumor blocks with and without PTEN deletion. These findings confirm that cancer foci with and without PTEN deletions may exist within the same prostate,
      • Han B.
      • Mehra R.
      • Lonigro R.J.
      • Wang L.
      • Suleman K.
      • Menon A.
      • Palanisamy N.
      • Tomlins S.A.
      • Chinnaiyan A.M.
      • Shah R.B.
      Fluorescence in situ hybridization study shows association of PTEN deletion with ERG rearrangement during prostate cancer progression.
      • Bismar T.A.
      • Yoshimoto M.
      • Vollmer R.T.
      • Duan Q.
      • Firszt M.
      • Corcos J.
      • Squire J.A.
      PTEN genomic deletion is an early event associated with ERG gene rearrangements in prostate cancer.
      but also demonstrate that it is unlikely that such heterogeneity becomes visible within an area of 0.6-mm cancer tissue analyzed per TMA spot. Our cutoff is substantially more stringent than in most previous FISH studies,
      • Han B.
      • Mehra R.
      • Lonigro R.J.
      • Wang L.
      • Suleman K.
      • Menon A.
      • Palanisamy N.
      • Tomlins S.A.
      • Chinnaiyan A.M.
      • Shah R.B.
      Fluorescence in situ hybridization study shows association of PTEN deletion with ERG rearrangement during prostate cancer progression.
      • Sircar K.
      • Yoshimoto M.
      • Monzon F.A.
      • Koumakpayi I.H.
      • Katz R.L.
      • Khanna A.
      • Alvarez K.
      • Chen G.
      • Darnel A.D.
      • Aprikian A.G.
      • Saad F.
      • Bismar T.A.
      • Squire J.A.
      PTEN genomic deletion is associated with p-Akt and AR signalling in poorer outcome, hormone refractory prostate cancer.
      • Yoshimoto M.
      • Cunha I.W.
      • Coudry R.A.
      • Fonseca F.P.
      • Torres C.H.
      • Soares F.A.
      • Squire J.A.
      FISH analysis of 107 prostate cancers shows that PTEN genomic deletion is associated with poor clinical outcome.
      • Reid A.H.
      • Attard G.
      • Ambroisine L.
      • Fisher G.
      • Kovacs G.
      • Brewer D.
      • Clark J.
      • Flohr P.
      • Edwards S.
      • Berney D.M.
      • Foster C.S.
      • Fletcher A.
      • Gerald W.L.
      • Moller H.
      • Reuter V.E.
      • Scardino P.T.
      • Cuzick J.
      • de Bono J.S.
      • Cooper C.S.
      Molecular characterisation of ERG, ETV1 and PTEN gene loci identifies patients at low and high risk of death from prostate cancer.
      in which the rate of artificial FISH signal losses caused by nuclei truncation was first determined in normal prostatic epithelium and then used as a threshold for deletion in cancer samples. In such a scenario, false deletion calling can occur because the larger nuclei of cancer cells will more often lose FISH signals because of truncation than the smaller normal cell nuclei. Accordingly, the highest frequencies of heterozygous deletion (44% to 59%) were reported from studies using less stringent thresholds (eg, ≥20% to 30% of tumor cells with FISH signal loss required to define deletion).
      • Sircar K.
      • Yoshimoto M.
      • Monzon F.A.
      • Koumakpayi I.H.
      • Katz R.L.
      • Khanna A.
      • Alvarez K.
      • Chen G.
      • Darnel A.D.
      • Aprikian A.G.
      • Saad F.
      • Bismar T.A.
      • Squire J.A.
      PTEN genomic deletion is associated with p-Akt and AR signalling in poorer outcome, hormone refractory prostate cancer.
      • Yoshimoto M.
      • Cunha I.W.
      • Coudry R.A.
      • Fonseca F.P.
      • Torres C.H.
      • Soares F.A.
      • Squire J.A.
      FISH analysis of 107 prostate cancers shows that PTEN genomic deletion is associated with poor clinical outcome.
      We found a slightly lower fraction of heterozygous deletions (8.1%) compared with homozygous deletions (12.1%). This is different from most previous studies
      • Han B.
      • Mehra R.
      • Lonigro R.J.
      • Wang L.
      • Suleman K.
      • Menon A.
      • Palanisamy N.
      • Tomlins S.A.
      • Chinnaiyan A.M.
      • Shah R.B.
      Fluorescence in situ hybridization study shows association of PTEN deletion with ERG rearrangement during prostate cancer progression.
      • Yoshimoto M.
      • Cutz J.C.
      • Nuin P.A.
      • Joshua A.M.
      • Bayani J.
      • Evans A.J.
      • Zielenska M.
      • Squire J.A.
      Interphase FISH analysis of PTEN in histologic sections shows genomic deletions in 68% of primary prostate cancer and 23% of high-grade prostatic intra-epithelial neoplasias.
      • Sircar K.
      • Yoshimoto M.
      • Monzon F.A.
      • Koumakpayi I.H.
      • Katz R.L.
      • Khanna A.
      • Alvarez K.
      • Chen G.
      • Darnel A.D.
      • Aprikian A.G.
      • Saad F.
      • Bismar T.A.
      • Squire J.A.
      PTEN genomic deletion is associated with p-Akt and AR signalling in poorer outcome, hormone refractory prostate cancer.
      • Verhagen P.C.
      • van Duijn P.W.
      • Hermans K.G.
      • Looijenga L.H.
      • van Gurp R.J.
      • Stoop H.
      • van der Kwast T.H.
      • Trapman J.
      The PTEN gene in locally progressive prostate cancer is preferentially inactivated by bi-allelic gene deletion.
      • Yoshimoto M.
      • Cunha I.W.
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      • Torres C.H.
      • Soares F.A.
      • Squire J.A.
      FISH analysis of 107 prostate cancers shows that PTEN genomic deletion is associated with poor clinical outcome.
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      • Yoshimoto M.
      • Vollmer R.T.
      • Duan Q.
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      • Squire J.A.
      PTEN genomic deletion is an early event associated with ERG gene rearrangements in prostate cancer.
      that usually reported more heterozygous (12% to 62%) than homozygous (5% to 25%) deletions. However, PTEN FISH results are highly variable in the literature, and some studies reported markedly higher rates of heterozygous (39% to 62%) compared with homozygous (5% to 6%) deletions,
      • Yoshimoto M.
      • Cutz J.C.
      • Nuin P.A.
      • Joshua A.M.
      • Bayani J.
      • Evans A.J.
      • Zielenska M.
      • Squire J.A.
      Interphase FISH analysis of PTEN in histologic sections shows genomic deletions in 68% of primary prostate cancer and 23% of high-grade prostatic intra-epithelial neoplasias.
      • Yoshimoto M.
      • Cunha I.W.
      • Coudry R.A.
      • Fonseca F.P.
      • Torres C.H.
      • Soares F.A.
      • Squire J.A.
      FISH analysis of 107 prostate cancers shows that PTEN genomic deletion is associated with poor clinical outcome.
      whereas others
      • Han B.
      • Mehra R.
      • Lonigro R.J.
      • Wang L.
      • Suleman K.
      • Menon A.
      • Palanisamy N.
      • Tomlins S.A.
      • Chinnaiyan A.M.
      • Shah R.B.
      Fluorescence in situ hybridization study shows association of PTEN deletion with ERG rearrangement during prostate cancer progression.
      • Sircar K.
      • Yoshimoto M.
      • Monzon F.A.
      • Koumakpayi I.H.
      • Katz R.L.
      • Khanna A.
      • Alvarez K.
      • Chen G.
      • Darnel A.D.
      • Aprikian A.G.
      • Saad F.
      • Bismar T.A.
      • Squire J.A.
      PTEN genomic deletion is associated with p-Akt and AR signalling in poorer outcome, hormone refractory prostate cancer.
      • Verhagen P.C.
      • van Duijn P.W.
      • Hermans K.G.
      • Looijenga L.H.
      • van Gurp R.J.
      • Stoop H.
      • van der Kwast T.H.
      • Trapman J.
      The PTEN gene in locally progressive prostate cancer is preferentially inactivated by bi-allelic gene deletion.
      found more similar frequencies of heterozygous (12% to 34%) and homozygous (9% to 25%) deletions. These differences may be related to different scoring criteria but also to the comparatively few samples and the presence or absence of hormone-refractory cancers in individual studies. The results of our analysis fit best to the findings reported by Han et al,
      • Han B.
      • Mehra R.
      • Lonigro R.J.
      • Wang L.
      • Suleman K.
      • Menon A.
      • Palanisamy N.
      • Tomlins S.A.
      • Chinnaiyan A.M.
      • Shah R.B.
      Fluorescence in situ hybridization study shows association of PTEN deletion with ERG rearrangement during prostate cancer progression.
      who applied a comparable definition for heterozygous deletion (>50% of tumor cells with signal loss), as used in our study. The authors reported 12.6% heterozygous and 9.2% homozygous deletions in a set of 251 hormone-naïve and 41 hormone-refractory cancers, which is close to the 8.1% heterozygous and 12.1% homozygous deletions found in our study.
      In summary, our data demonstrate that PTEN deletions are found in approximately 20% of prostate cancers, and represent a major driver of patient prognosis independent of ERG status. The frequent finding of homozygous deletions or combinations of a heterozygous deletion and mutation, in two thirds of PTEN-defective cancers, suggests a strong selective advantage for tumor cell clones with complete PTEN inactivation. These tumors account for approximately 15% of all prostate cancers and are characterized by particularly aggressive features, including hormone-independent and metastatic growth.

      Acknowledgments

      We thank Silvia Schnöger and Sascha Eghtessadi for their excellent technical support.

      Supplementary data

      • Supplemental Figure S3

        Examples of PTEN immunostaining results in prostate cancers with different PTEN copy number status (normal, 2 copies; heterozygous deletion, 1 copy; homozygeous deletion, complete lack of PTEN gene copies). A: Overview of a small TMA immunostained with antibody (Abcam ab31392) at two different dilutions. B: Magnification of the boxed areas. Nuclear staining is seen in all tissue spots irrespective of PTEN copy number status.

      • Supplemental Figure S4

        Examples of PTEN mutations. A: c.1067_1070del (p.Asn12_LysfsX11) 4-bp deletion in exon 1. B: c.352C>T (p.H118Y) mutation in exon 5. C: c.1632G>T (p.E201X) mutation in exon 6. D: c.1981_1984del (p.Val317_ValfsX2) 4-bp deletion in exon 1. E: c2007G>A (p.D326N) mutation in exon 8. The PTEN wild-type DNA sequence is shown below each case.

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