Which, when and why? Rational use of tissue-based molecular testing in localized prostate cancer

Abstract

An increased molecular understanding of localized prostate cancer and the improved ability for molecular testing of pathologic tissue has led to the development of multiple clinical assays. Here we review the relevant molecular biology of localized prostate cancer, currently available tissue-based tests and describe which is best supported for use in various clinical scenarios. Literature regarding testing of human prostate cancer tissue with Ki-67, PTEN (by immunohistochemistry (IHC) or fluroescence in situ hybridization (FISH)), ProMark, Prolaris, OncotypeDX Prostate and Decipher was reviewed to allow for generation of expert opinions.

At diagnosis, evaluation of PTEN status, use of ProMark or OncotypeDX Prostate in men with Gleason 6 or 3+4=7 disease may help guide the use of active surveillance. For men with Gleason 7 or above disease considering watchful waiting, Ki-67 and Prolaris add independent prognostic information. For those men who have undergone prostatectomy and have adverse pathology, Decipher testing may aid in the decision to undergo adjuvant radiation. Newly available molecular tests bring opportunities to improve decision making for men with localized prostate cancer. A review of the currently available data suggests clinical scenarios for which each of these tests may have the greatest utility.


Introduction
Localized prostate cancer represents a disease spectrum, with ideal treatment varying from watchful waiting (WW) to immediate multi-modal therapy. Although clinical and pathological features of disease can guide decision making, there remains ambiguity even among risk-stratified patients. An increased molecular understanding of localized prostate cancer and facility for molecular testing has led to the development of tissue-based assays that may help decrease the uncertainty in decision making. Because many of these tests occupy overlapping clinical spaces, assess similar molecular phenotypes and have aggressive marketing, there can be confusion over which test to choose in a given clinical scenario. Here we briefly review the molecular biology of localized prostate cancer and provide a framework for the use of these clinical tests.

Relevant molecular biology of localized prostate cancer
Through genomic analysis, in vitro and mouse model experiments, the molecular underpinnings of localized prostate cancer are being elucidated. Notable early events include the inactivation of tumor suppressors, specifically PTEN, and dysregulation of the cell cycle. In addition, a common event involves rearrangement between androgen responsive genes (such as TMPRSS2) and a member of the ETS transcription factor family (most commonly ERG).

PTEN is a tumor suppressor gene residing on chromosome 10q.1 It normally functions to halt the PI3K/AKT pathway which drives cellular growth, survival, motility and polarity.1 The PI3K signaling pathway is disrupted in up to about 40% of localized prostate cancer cases and virtually 100% of metastatic men with PTEN expression loss itself occurring in 15–40% of primary cases (with the frequency of loss increasing with stage and Gleason grade).2, 3, 4 Loss of PTEN expression can occur via multiple mechanisms, but most commonly occurs via chromosomal loss. Although other tumor suppressors (such as NKX3.1) are also commonly lost early in carcinogenesis, PTEN loss appears to be a critical biological point in the evolution of prostate cancer with metastatic potential.5 Physiologic evidence supporting this comes from animal models, where prostate-specific PTEN loss results in the development of adenocarcinoma which becomes locally advanced and metastatic when combined with additional oncogenic factors (such as Myc expression) or tumor suppressor loss (such as disruption of SMAD4/ the TGF beta pathway).6, 7

Genes affecting the cell cycle are frequently deregulated in prostate cancers. Specifically, alterations in CDKN1B (p27) and TP53 and dysregulation of their downstream effectors is commonly seen.4 Of note, the pathologic mitotic rate and overall proliferative index of prostate cancer is low, and this may be reflective of the long natural history of localized disease.

In regards to ERG expression, as mentioned above, chromosomal rearrangements involving the 5′-untranslated region of androgen responsive genes and ETS family members occur in ~50% of localized prostate cancer.bib88 These rearrangements may be related to transcriptional instability at androgen responsive genes and ERG status might be reflective of androgen signaling.bib99 In addition to its role as a transcription factor, ERG may have a cytosolic role in microtubule regulation.10

Materials and methods
PubMed was queried for all publications regarding human prostate cancer tissue testing with for either Ki-67 (MIB-1), PTEN (immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH)), ProMark, Prolaris, OncotypeDX Prostate and Decipher. All publications were examined with only literature involving the testing of formalin-fixed paraffin-embedded (FFPE) tissue reviewed. Salient and clinically relevant findings from these publications were reported. Where available, odds ratios and hazard ratios were reported from mutli-variable analysis.

Results
Currently, multiple tests are available, all of which can be performed on routinely stored FFPE tissue. Below and in Table 1, we summarize the literature regarding these tests.



Tests based on cell cycle proliferation
Cell proliferation in cancer has traditionally been quantified by semi-quantitative IHC of Ki-67, a nuclear protein associated with ribosomal RNA synthesis. In addition, a quantitative RT-PCR assay known as Prolaris, which measures 31 cell-proliferation markers, has been developed by Myriad (Salt Lake City, UT, USA) for use in biopsy and prostatectomy tissue.

Ki-67 IHC
Typically Ki-67 IHC is performed using a MIB-1 antibody and reported as the percent of cells staining positively for Ki-67. Ki-67 staining has been studied in men managed by radiation or surgery for prostate cancer, as well as in those undergoing conservative management.

  • In RTOG 92-02, the pretreatment biopsies of patients undergoing therapy with androgen deprivation and radiation were examined.11 A dichotomous cutoff for Ki-67 staining was set at 7.1%. Ki-67 staining>7.1% significantly correlated with distant metastasis (mets) and prostate cancer-specific mortality. Ki-67 staining also had prognostic power as a continuous variable on multivariable analysis (MVA) with data suggesting that higher cutoff points might be more informative. In a follow-up study, Ki-67 staining of 11.3% correlated with hazard ratios (HR) of distant mets, cancer-specific death and overall death were 2.95, 2.35 and 1.44, respectively.12
  • Ki-67 IHC has been examined in pretreatment biopsies prior to prostatectomy, as well as in radical prostatectomy (RP) tissue. Ki-67 staining in pretreatment biopsies prior to RP was explored in a large series of 445 men from the Mayo Clinic.13 A dichotomous cutoff of 6% positive staining was used. Eleven percent of men had high Ki-67 staining, and had increased risks of mets and cancer-specific mortality. Ki-67 staining was an independent predictor and added value to D’Amico and Kattan nomograms. When analyzed on surgical specimens Ki-67 has also showed prognostic ability for cancer-specific outcomes with HRs of 1.5 to 4.4 in MVA analyses (summarized by Fischer et al. \14). The range of HRs is likely reflective of a lack of a uniform cutoff and quantitation method, as well as differences in the overall risk of the studied populations.
  • Ki-67 has been studied in untreated men, such as men from the Transatlantic Prostate Group. This cohort contained 243 analyzable cases of men diagnosed prior to the widespread use of PSA screening in Great Britain (1990–1996) who had long clinical follow-up (median of 9 years) and who were managed by WW.14 A cutoff of >10% was used for positive staining. Only 5% of the population had high Ki-67 staining on their biopsies, and these men were at higher risk for prostate cancer mortality (HR of 2.8 on MVA). Only one man with a Gleason sum of 6 had >10% Ki-67 staining.
Prolaris
The Prolaris test can be run on biopsy or prostatectomy tissue and involves quantitative RT-PCR of 31 cell cycle-related genes and 15 housekeeping genes. The assay was developed by Myriad and reports a proliferative index, expressed as a cell cycle progression (CCP) score. Like Ki-67, the CCP score has been tested in multiple clinical cohorts with known outcomes.15, 16, 17, 18 CCP has CMS approval for use on biopsy tissue from low and very-low risk men to help determine if immediate or deferred treatment should be pursued.

  • CCP scores were analyzed, like Ki-67, in needle biopsies from the Transatlantic Prostate Group of conservatively managed patients.17 Of 349 analyzable men, median CCP score was 1.03 (interquartile range 0.41–1.74). The cumulative incidence of death was increased among those with CCP scores >2 (19% of the population). The cumulative incidence of death was similar among men in lower score categories (CCP scores of 1 to 2, 0 to 1, or <0). On MVA, the HR of prostate cancer death was 1.7 per unit increase in CCP score (representing a doubling in cell cycle gene expression). As with Ki-67, very few patients with Gleason 6 disease had CCP scores of 2 or greater, and CCP score was not predictive of outcome among men with Gleason 6 disease (HR 1.3, 95% CI 0.61–2.77).
  • CCP scores from biopsy tissue were also evaluated in men undergoing RP. In a multi-institutional retrospective study of men undergoing surgery, CCP was found to be independently associated with biochemical recurrence (BCR) and mets (HR 1.47 and 4.19, per unit score increase; P<0.05 for both).15 There were relatively few cases of mets in this cohort, again with men having biopsy CCP scores >2 (2.4% of the study population) having the worst metastatic outcome and men having lower CCP scores all having similar and low rates of mets. In a separate study on biopsy tissue of men undergoing radiation therapy, CCP score increases were also associated with biochemical failure (Phoenix definition), with a HR of 2.1 per unit increase in score (P=0.03) on MVA.18 Here, a separation among Kaplan–Meier curves were seen for those patients with CCP scores <0 compared with those with CCP scores >0. Very few men experienced cancer-specific death or mets in this cohort and CCP could not be evaluated in a MVA model for these outcomes.
Tests based on molecular characteristics of prostate cancer

PTEN testing
PTEN loss can be measured on FFPE tissue at the DNA level by FISH (such as is performed in the ProstaVysion test) and at the protein expression level through IHC. The Transatlantic Prostate Group has performed a head-to-head evaluation of PTEN loss by both IHC and FISH. Performance characteristics of PTEN testing in this comparison were most favorable when PTEN was evaluated by IHC (perhaps because IHC can detect loss of PTEN protein expression whether due to gene silencing or chromosomal loss).

  • Evidence regarding PTEN loss in conservatively managed patients comes from the Transatlantic Prostate Group.19 Here, among 675 men, 18% demonstrated PTEN loss by IHC. PTEN loss was highly predictive of prostate cancer mortality in men with low-risk characteristics (those with low Gleason score, low PSA, low Ki-67 staining and low extent of disease) with a HR of prostate cancer death of 7.4, P=0.012. Among those with Gleason sum<7, PTEN loss had a HR of 8.13 (CI 2.8–23.2). Although PTEN loss in a low-risk man lead to cancer-specific survival similar to a high-risk man, only 3% of the low-risk cohort had PTEN loss. Also, PTEN loss did not correlate with prognosis in high-risk men. A limitation of this work was that evaluation of PTEN was performed on transurethral resection specimens and thus may not be directly translatable to a needle biopsy cohort.
  • PTEN loss has been evaluated in needle biopsy cohorts of men undergoing RP using IHC. In one study, 174 men diagnosed with Gleason 6 disease who underwent prostatectomy were evaluated.20 PTEN loss was observed in 11%. On MVA analysis, PTEN loss on biopsy was predictive of upgrading at surgery (odds ratio 3 (95% CI 1.1–8.6)). In another study, PTEN loss was interrogated in biopsies of 77 men undergoing RP by the PREZEON assay.21 Twelve percent of men had PTEN loss, with 77% of those men having Gleason scores of 7 or higher. PTEN loss was not significantly associated with BCR on primary analysis. In secondary, univariate analysis, PTEN loss significantly associated with the development of mets, castrate resistant prostate cancer and death (with no men without PTEN loss developing metastatic disease).
  • PTEN has been examined in prostatectomy tissue. In a series of 217 patients studied by PTEN IHC, PTEN loss was a significant determinant of future mets after prostatectomy in UVA, but not in models incorporating stage, grade and surgical margin status.22 This may be reflective of the higher frequency of PTEN loss among men with higher Gleason score and pathologic stage. In other studies however, PTEN loss on prostatectomy tissue has been shown to be predictive of poor outcomes. For instance, PTEN homozygous loss has also been shown to be independently predictive of BCR when assayed by FISH in RP series that are at high risk of disease progression.22,23 In addition, in men participating in the adjuvant docetaxel TAX2501 trial, PTEN loss in the RP specimen was an independent predictor of disease progression.24
ProMark
ProMark was developed by Metamark (Cambridge, MA, USA) as a quantitative multiplexed proteomics assay. After assessing 160 candidate markers 12 were identified that predicted prostate cancer aggressiveness by pathology and clinical outcomes despite biopsy sampling error.25,26 This signature was refined to include 8 of the 12 protein markers which were then validated on 276 matched prostate biopsy and RP specimens for their ability to predict unfavorable or high grade pathology at RP.27 The test outputs a score between 0 and 1 and, on multivariate analysis, the odds ratio for unfavorable pathology for each 0.25 shift in score was 3.1 (P<0.001). Thirty nine percent of their cohort had scores that were <0.33 or >0.8. At these thresholds the test had a sensitivity of 90% and specificity of 95%, respectively.

OncotypeDX Prostate
Like Prolaris, this is a quantitative RT-PCR assay performed on FFPE tissue from needle biopsies. The assay, developed by Genomic Health (Redwood City, CA, USA), incorporates 12 cancer genes involved in 4 biological pathways, including the androgen receptor pathway, proliferation, cellular organization and stromal response with 5 reference genes. The assay was developed using RP and biopsy specimens and outputs a genomic prostate score (GPS) which ranges from 0 to 100 (ref. bib2828). The test is marketed for use on biopsy tissues from men with low-risk or low intermediate risk prostate cancer considering active surveillance (AS).

  • Use of OncotypeDX Prostate has not yet been reported in populations of men undergoing AS or WW with clinical follow-up.
  • OncotypeDX Prostate has been examined in large cohorts of men undergoing RP. A validation study was performed including 395 men who had low-risk or low intermediate-risk prostate cancer and underwent prostatectomy.28 The primary end point was adverse pathology at prostatectomy (high grade (primary pattern 4 or greater or any pattern 5, or non-organ confined disease)). Here, each 20-point increase (roughly the interquartile range of the cohort) in GPS was associated with a statistically significant increased risk of adverse pathology at prostatectomy (odds ratio ~2.1, (95% CI 1.4–3.2) in a MVA model with CAPRA). In a second study, biopsies from 402 men with low or intermediate-risk prostate cancer were analyzed for the ability of GPS to predict adverse pathology and BCR after RP.29 On MVA, GPS was independently associated with adverse pathology (odds ratio 2.7 (95% CI 1.77–4.36). In this study however, GPS was not compared to CAPRA which stratifies PSAs (including those under 10) and contains information regarding percent biopsy core involvement. In regards to clinical outcomes after treatment, 15% experienced BCR and 1% experienced mets with a median follow-up of 5.2 years. On UVA, each 20-point GPS score increase correlated to a HR of 2.9, P<0.001 or 3.8, P=0.03 for the development of BCR or mets, respectively. GPS was not compared with posttreatment nomograms or clinico-pathologic features in this analysis, but on MVA analysis with pre-operative features it was independently predictive of BCR. MVA analysis could not be performed for mets given the low event rate.
Decipher
Unlike the previously mentioned RNA-expression assays, Decipher was developed and is performed by GenomeDX Biosciences as a CLIA-certified high-density microarray, analyzing 1.4 million genomic markers including coding and non-coding RNAs.30 Decipher is performed using routinely stored FFPE tissue. The test reports a Decipher mets signature as a genomic classifier score (GC) ranging from 0 to 1. This signature is based on 22 markers which relate to cell proliferation, differentiation, androgen signaling, motility and immune modulation. Decipher was developed on RP tissue with a benchmark of predicting rapid metastatic progression. Currently, Decipher is CMS approved for use in post-prostatectomy decision making. Decipher has been studied in post-RP cohorts, including ones receiving, and not receiving additional therapy prior to metastatic progression.

  • In a validation study, the Decipher mets signature was evaluated on 219 high-risk men from the Mayo Clinic undergoing RP.31 With a median follow-up of 6.7 years, 32% experienced mets. On MVA, higher GC scores were independently predictive for the development of mets (HR of 1.5 for each 10% increase (0.1) in score). As a categorical variable, GC between 0.4 and 0.6 (21% of the cohort) or >0.6 (19% of the cohort) were associated with HRs for mets of 2.4 (95% CI 1.1–5.2) and 7.3 (95% CI 3.5–15.1) compared GC scores <0.4. Though a substantial proportion of this cohort had received adjuvant or salvage therapy prior to mets, GC remained predictive in sub-set analysis of men treated only with prostatectomy prior to clinical progression.
  • Decipher was also evaluated in 169 men treated by RP at the Cleveland Clinic.bib3232 These men had a median follow-up of 8 years and did not receive adjuvant therapy. Twenty nine percent of the cohort developed mets with 9% developing mets within 5 years of surgery. Median GC was 0.35 (interquartile range 0.2–0.6) and was increased in men who developed rapid (<5 years) mets (OR 1.5 (95% CI 1.1–2.1) for each 10% increase in score on MVA).
  • Decipher was also evaluated in a cohort of 260 NCCN intermediate and high-risk men from Johns Hopkins, who underwent RP and did not receive any adjuvant or salvage therapy until the time of mets.33 Of these, 99 (38%) developed mets with a median follow-up of 9 years. Median GC score was 0.34 (interquartile range 0.22–0.52). On MVA for development of mets, GC had a HR of 1.5 (95% CI 1.3–1.7) per 10% increase in score. The Decipher mets signature had the highest c-index (0.76) for prediction of metastatic outcome when compared to 33 other previously published gene signatures.
  • Decipher has been explored in populations of men receiving adjuvant or salvage radiation therapy following prostatectomy.bib3434, bib3535 In one study, 188 men from Thomas Jefferson University and the Mayo Clinic who had undergone prostatectomy, had adverse pathologic features, and had either adjuvant or salvage radiation were evaluated.35 Median follow-up was 10 years. GC was predictive of mets on MVA (HR 1.9; 95% CI 1.3–2.8). Importantly, for men with low GC scores (<0.4) the mets rate was low and similar among men receiving either adjuvant or salvage radiation therapy. In contrast, among men with high GC scores (0.4) the mets rate at 5 years was 6% for those treated with adjuvant therapy versus 23% for those men receiving salvage radiation (P<0.008).
  • In an exploratory study, 85 men with BCR after prostatectomy were evaluated.36 Among the 85 men studied, GC>0.4 was associated with poor outcomes (40% metastatic progression versus 8% in those with GC<0.4). In MVA, only PSA doubling time and the GC were statistically significant predictors of mets after BCR (HR of 1.5 for every 10% increase in GC).
Discussion
Upon diagnosis with localized prostate cancer, the initial clinical decision facing a man is regarding whether or not any treatment is necessary. For those where treatment is selected, some may require multi-modal therapy. On the basis of the above synthesis of the literature we make recommendations for which tests may be preferred in the various clinical scenarios (Table 2).



Clinical decision making regarding surveillance—PTEN IHC, ProMark, OncotypeDX Prostate
Strong evidence supports the use of AS in men with very-low risk and low-risk disease. In two randomized clinical trials regarding RP or WW, men over 65 with low-risk disease had minimal benefit to local intervention.37,38 Further, robust AS cohorts report low rates of adverse oncologic outcomes when AS is chosen for very-low and low-risk men.39, 40, 41 Of note however, low-risk men have a higher disease risk re-classification rate over time, and AS populations with long-term follow-up have been comprised primarily of older men.40

On the basis of the above data, we do not recommend the routine use of ancillary molecular testing for men over 65 years with very-low risk prostate cancer, and these men should be encouraged to undergo AS. Molecular testing may benefit younger men (and those with >20 year life expectancy), low-risk men and likely men with low-intermediate-risk men considering AS (though evidence is limited for this later group). Among these men, the current evidence most strongly supports PTEN, ProMark or OncotypeDX Prostate testing with the expectation that testing will be confirmatory (or un-informative) in most. Cell cycle proliferation tests (Ki-67 and Prolaris) appeared un-informative among men with Gleason 3+3=6 disease. Importantly, results of OncotypeDX prostate and ProMark have not yet been reported in men managed without treatment, unlike PTEN which has been studied in both WW and prostatectomy cohorts. It is also important to note that cancer is not static, and serial testing on biopsy tissue would be warranted in men on AS. As this is the case, PTEN IHC may be further preferred since its cost is far lower than RNA-expression based tests ($96.00 at the first author’s institution compared with $3400.00 for Prolaris and $3825.00 for OncotypeDX Prostate)42 and will likely be lower than ProMark.

Tests and clinical decision making regarding WW—Ki-67 IHC, Prolaris
For men with limited life expectancy, WW is best supported by evidence. Use of WW is limited and can be anxiety provoking for patients and treating physicians, particularly for men diagnosed with intermediate and high-risk localized prostate cancer, as there may be benefit for local therapy even if survival is over 5 years.bib3838 When considering WW, the above evidence most strongly supports the use of a proliferative index. Both Ki-67 IHC and Prolaris appear to function similarly, but head-to-head comparisons have not been performed. Ki-67 staining, like PTEN IHC is less expensive; however, heterogeneity in Ki-67 staining across labs might limit its clinical utility compared with Prolaris.43

Tests and clinical decision making regarding the aggressiveness of primary radiation—needs study
For those men who undergo local therapy for prostate cancer, RP and radiation therapy have the best evidence for oncologic control. As opposed to RP, radiation is biologic and is modulated in its delivery based on predictive cancer aggressiveness. Though clinical trials are ongoing, controversy exists as to whether all intermediate-risk men may benefit from adjuvant hormonal suppression.44 In addition, not all high-risk men benefit from long-term androgen deprivation. Finally, some men may be less sensitive to radiation based on the molecular characteristics of their tumor. Though Prolaris and Ki-67 have both been studied in cohorts undergoing primary radiation therapy, no study has directly attempted to answer the above clinical questions and the authors cannot make recommendations here.

Tests and clinical decision making regarding adjuvant therapy after treatment—Decipher
Currently, ASCO, ASTRO and the AUA all recommend that adjuvant radiation therapy should be discussed with/men having adverse pathological features (pT3 disease or positive surgical margins) at RP.45, 46 Not all men with adverse pathologic features will benefit however. Results from SWOG 8794 trial suggest a number needed to treat of ~12 men to prevent one metastatic outcome while EORTC 22911 did not show improvements in overall or prostate-specific survival among men receiving adjuvant versus a wait and see approach with >10-year median follow-up.47, 48 Although a randomized controlled trial of adjuvant versus early salvage radiation therapy is underway, current decision making to undergo adjuvant radiation therapy can be challenging, particularly when considering toxicity of treatment.49 In this clinical space, the above evidence most strongly supports use of the Decipher mets signature to help identify men who would most benefit from adjuvant radiation therapy and might miss a window of opportunity if observed until PSA recurrence.

Conclusions
The availability of molecular testing may improve clinical decision making in localized prostate cancer. Although studies examining whether these molecular tests can change clinical decision making have been reported and are underway,50,51 these have not been linked to disease outcomes and thus the true clinical value of these tests, remains to be established through prospective registries and clinical trials (Apolo et al.52 provides a good representation of such a study). Though the indications of molecular tests will likely be refined with further study and development, these tests are currently available and have retrospective evidence supporting their utility. Here we have reviewed the evidence of available molecular tests and put their usage into clinical context and made recommendations to help providers and patients know which tests might be appropriate and when they should consider their use.

Conflict of interest
AER has received compensation for consultation from GenomeDx Biosciences (manufacturers of Decipher). SJF has performed collaborative research with Myriad (manufacturers of Prolaris) and GenomeDx Biosciences. AVD declares no conflict of interest.

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Acknowledgements
AER is funded by the Johns Hopkins Clinician Scientist Award, Patrick Walsh Research Fund and the DOD Prostate Cancer Physician Research Training Award.

Written by: A E Ross1,2,3, A V D'Amico4 and S J Freedland5
1
Department of Urology, James Buchanan Brady Urological Institute, Johns Hopkins Medical Institutions, Baltimore, MD, USA
2Department of Oncology, James Buchanan Brady Urological Institute, Johns Hopkins Medical Institutions, Baltimore, MD, USA
3Department of Pathology, James Buchanan Brady Urological Institute, Johns Hopkins Medical Institutions, Baltimore, MD, USA
4Department of Radiation Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA
5Division of Urology, Department of Surgery, Cedars-Siani Medical Center, Los Angeles, CA, USA

Correspondence: Dr AE Ross, James Buchanan Brady Urological Institute, Johns Hopkins Medical Institutions, Marburg 134, 600 North Wolfe Street, Baltimore, MD 21287, USA. E-mail: 

Prostate Cancer and Prostatic Disease (2016) 19, 1–6; doi:10.1038/pcan.2015.31; published online 30 June 2015

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