Protein Expression of PTEN, Insulin-Like Growth Factor I Receptor (IGF-IR), and Lethal Prostate Cancer: A Prospective Study

The use of prostate-specific antigen (PSA) screening for prostate cancer has likely contributed to reductions in cancer-specific mortality (1, 2), while at the same time, overtreatment is a concern for many PSA test–detected cancers (3, 4). The reason for such a controversy lies in the heterogeneous nature of prostate cancer, of which the clinical behaviors vary greatly (5–8). While many tumors grow slowly and would not pose a threat if left untreated, some clinically organ-confined tumors progress rapidly and develop metastasis even after local therapy. Therefore, it is of urgent need and great clinical importance to identify key factors or molecular events that can determine or predict the behaviors of the tumor.

One such marker for prostate cancer is phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumor suppressor that negatively regulates the phosphoinositide-3-kinase (PI3K)/Akt pathway (9, 10). Loss or inactivation of PTEN, usually leading to constitutive activation of the PI3K/Akt pathway, has been implicated in a number of human malignancies including prostate cancer (11–13). In animal models, prostate-specific PTEN knockout can lead to tumorigenesis, progression, and metastasis of prostate cancer (14–16). In epidemiologic and clinical studies, loss or reduction of PTEN in prostate tumors has been shown to be associated with higher grade (17–20), more advanced stages (18, 20), greater angiogenesis (21), faster progression to androgen independence (22), and increased risk of recurrent disease (22–25). The role of PTEN loss in association with prostate cancer–specific mortality, however, is less clear. Both positive and null findings have been reported (18, 19, 26). Homozygous deletions or mutations of PTEN gene, resulting in total loss of PTEN activity, occur at relatively low frequencies of 10% to 15% in primary prostate cancer (11, 21). However, a significantly higher proportion of tumors has reduced PTEN protein level (17, 20, 27), and such reductions may be sufficient to influence prostate cancer survival (24, 26).

One of the activators of the PI3K/Akt pathway is the insulin-like growth factor 1 (IGF1; refs. 28, 29). Initiation of downstream signaling by IGF1 is predominantly mediated by the IGF-I receptor (IGF-IR), which is ubiquitously expressed across different tissues, promotes cell proliferation, differentiation, and inhibits apoptosis. Overexpression or constitutive activation of IGF-IR is shown to promote tumor formation in mouse models (30, 31). Several clinical studies reported that high IGF-IR expression has been detected in human tumor specimens and the expression level correlates with metastasis (32–34). Epidemiologic studies on IGF1 signaling in relation to risk of prostate cancer have nearly exclusively focused on circulating IGF1 and IGF-binding proteins (35–46). Although some recent findings have been null, the majority of the studies support a positive association between IGF1 and prostate cancer risk.

We hypothesize that reduction or loss of PTEN expression in prostate tumor contributes to cancer progression. As the PI3K/PTEN/Akt pathway is downstream of IGF1/IGF-IR, the effect of PTEN loss may be modified by IGF1 signaling. To test our hypotheses, we examined tumor protein expression of PTEN and IGF-IR from specimens of 805 men who were diagnosed with prostate cancer and underwent radical prostatectomy as therapy and followed them prospectively for cancer-specific mortality or development of distant metastasis. We evaluated whether PTEN protein expression in tumor is related to the risk of lethal prostate cancer, defined by cancer-specific mortality or distant metastasis. We also investigated IGF-IR expression in tumor as a potential effect modifier for the association between PTEN expression and lethal prostate cancer risk. The research was approved by the Institutional Review Board at the Harvard School of Public Health and the Partners Health Care.

The men with prostate cancer in this study were participants in the prospective Physicians' Health Study (PHS) and Health Professionals Follow-up Study (HPFS). The PHS was a randomized trial among 29,067 U.S. male physicians ages 40–84 years at randomization in the primary prevention of cancer and cardiovascular disease. Participants are sent annual questionnaires to ascertain disease endpoints as well as lifestyle, dietary, and medical covariate data as part of the main trial. The HPFS is an ongoing prospective cohort study of 51,529 U.S. male health professionals who were aged 40 to 75 years at baseline in 1986. The participants have been sent a questionnaire every 2 years since 1986 to update their information on lifestyle factors, medical history, and disease outcomes.

The PHS and HPFS Prostate Tumor Cohort is a sample of participants for whom we had retrieved archival formalin-fixed, paraffin-embedded prostatectomy (95%) and TURP (5%) tumor specimens from these men. The current analysis is based among 804 men (329 from PHS and 475 from HPFS) for whom the first batch of molecular assessment of PTEN expression was completed.

In both the PHS and HPFS cohorts, cases of prostate cancer were identified by self-report, then confirmed by review of medical records and pathology reports. Clinical information, such as tumor stage and PSA at diagnosis, was acquired through a standardized review of medical records. The cohorts were followed prospectively for cancer and all-cause mortality. Deaths were ascertained through repeated mailings, telephone calls to nonrespondents, and searches of the National Death Index. All causes of death were confirmed by extensive review of death certificates and medical records. Follow-up for cancer was >96% complete and for mortality >98% complete. Participants who had prostate cancer were separately followed. Detailed information on treatment and development of metastasis was obtained via additional questionnaires and collection of medical records.

Protein expression of endothelial cell marker CD34 was ascertained on 5 micron sections using the anti-CD34 mouse monoclonal antibody (QBE ND 10, BioGenex, CA, diluted 1:200) and peroxidase blocking reagent (Dual Endogenous Enzyme Block, DakoCytomation). Detection was accomplished by a peroxidase-labeled polymer conjugated to anti-mouse IgG (DakoCytomation), liquid 3,3′-diaminobenzidine (DAB)+ Substrate Chromogen System (K3468), and DakoCytomation EnVision+Dual Link System (DakoCytomation). Immunohistochemistry was conducted in an OptiMax automated cell staining system (BioGenex Laboratories). Slides were counterstained with hematoxylin (Sigma-Aldrich). The size and architecture of microvessels were quantified by semi-automated image analysis using Image ProPlus 4.5 software (Media Cybernetics). Vessel size was determined as the average area comprised by a vessel (μm²). The irregularity of the vessel lumen was calculated by perimeter²/4 × π × area, with a value of 1.0 indicating a perfect circle and values greater than 1.0 indicating increasing irregularity.

To increase efficiency of immunohistochemical staining, we constructed high-density tumor tissue microarrays (TMA). Our pathologists (M. Loda, M. Fiorentino, S. Finn, R. Flavin) reviewed hematoxylin and eosin slides to confirm prostate cancer and to provide uniform Gleason grading (47). TMAs were constructed by sampling 0.6-mm paraffin-embedded tissue cores (at least 3 cores per subject) and embedding the cores on a recipient array block.

Cytoplasmic expression of PTEN in tumor cells was ascertained using the Anti-PTEN rabbit polyclonal antibody (Zymed; diluted 1:200) and was quantified using automated image analysis. Briefly, 4-μm sections of each TMA were mounted on a glass slide, deparaffinized, and microwaved for antigen retrieval in citrate-based buffer. Hematoxylin was used as a counterstain and 3,3′-diaminobenzidine (DAB) for the immunohistochemical (IHC) staining. The stained slides were then scanned using the Ariol Pathology and Imaging System (Applied Imaging), and PTEN expression was quantified using the Ariol Immunohistochemical Module. Following image acquisition of the stained slide, a custom mask was made for each core to limit analysis to tumor cells. The IHC module in Ariol outputs information on the staining intensity for a given core (range, 0–255, mean and mode intensity across core), the area of DAB staining and blue counterstain which allows calculation of percent of tumor tissue staining positive for PTEN (0%–100%).

IHC staining was conducted on 5-μm sections of each TMA using the Anti-Insulin Receptor, B subunit, Rabbit immunoaffinity-purified IgG (Upstate Cell Signaling Solutions) and the IGF-1R beta R rabbit polyclonal antibody (Santa Cruz Biotechnology Inc.CA) with enzyme-labeled biotin streptavidin system and solvent-resistant DAB Map kit (Ventana autostainer model Discover XT, Ventana Medical System). Nonspecific reactivity was assessed by omission of the primary antibody. The specificity of staining for IR was confirmed by using placenta as a positive control. The slide was scanned with the BLISS system (Bacus Lab) and scored manually by 2 pathologists who were blinded to clinical outcomes. For both receptors, intensity was scored from 0 to +3—0 (no staining by any tumor cells), 1 (faint or focal staining), 2 (moderate intensity in a convincing number of cells), and 3 (intense staining by a sufficient number of cells).

The terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) assay was used on 5-μm sections to identify the percentage of tumor cells undergoing apoptosis. The procedure was carried out using the Apoptag Peroxidase In situ Kit (Chemicon International) according to the manufacturer's instructions. The entire area of each tumor core was evaluated. Apoptosis was quantified as the percentage of positively stained area over the whole tumor area.

The expression of Ki-67, a cell proliferation marker, was assessed on 5-μ sections using a rabbit polyclonal antibody (Vector Labs; diluted 1:1,500). After IHC staining, the tumor areas of each core were selected for quantitative image analysis using the Ariol instrument SL-50 (Applied Imaging). Cell proliferation was quantified as the percentage of Ki-67–positive nuclei over all tumor nuclei.

In the analyses presented here, we used the baseline questionnaire data when the participants entered the original HPFS and PHS studies as baseline for demographic measures.

For each individual, we calculated the minimum, mean, and maximum values for percent area staining and staining intensity of PTEN across the TMA cores for an individual. These values were qualitatively similar; therefore we only presented the analyses using the mean values. We consider the percent area staining, reflecting the percent of tumor tissue with PTEN expression, as the most relevant measure to evaluate the potential role of PTEN in cancer progression. In addition, we also generated a combined staining score, by multiplying the mean percent staining and the mean staining intensity, to reflect total PTEN expression in the tumor. These 2 measures were used as continuous variables in the analyses. They were also divided into quartiles according to the cohort distribution. In addition, we created an arbitrary cutoff point for each of these 2 measures by combining the bottom 3 quartiles as the low-expression group. Less than 32% is considered as low percent staining, and less than 32.25 as low score.

We compared clinical characteristics of prostate cancer cases including age at diagnosis, baseline body mass index (BMI), tumor stage, Gleason grade, and PSA at diagnosis, as well as cellular biomarkers for characteristics of angiogenesis (microvessel size and irregularity), apoptosis, and cell proliferation, across quartiles of PTEN expression. We calculated a P_trend value from ANOVA for age at diagnosis, baseline BMI, angiogenic characteristics, apoptosis, and cell proliferation across PTEN quartiles. Using the Mantel–Haenszel statistic, we tested whether there is a linear trend for tumor stage, Gleason grade, or PSA at diagnosis across quartiles of PTEN expression.

We used time-to-event analyses to evaluate the association of PTEN expression on development of lethal prostate cancer (cancer-specific death or development of distant metastasis). Cases were diagnosed with prostate cancer between 1983–2005 in PHS and 1986–2002 in HPFS. Person-time for each individual was calculated from the date of cancer diagnosis to development of metastases, death, or end of follow-up (July 1, 2010 for the HPFS and March 1, 2010 for the PHS). Hazard ratios and 95% confidence intervals (CI) were estimated from the Cox proportional hazard regression models. We adjusted for age at diagnosis (5-year categories), diagnosis era (pre-PSA era: before 1989, peri-PSA era: 1989–1993, PSA era: after 1993), baseline BMI (kg/m², continuous), and baseline smoking status (current, non-current). In additional models, we examined PTEN expression and lethal prostate cancer independent of tumor stage (T1/T2, T3, T4), Gleason score (6 or less, 7–10), and PSA at diagnosis (ng/mL; <4.1, 4.1–9.9, ≥10, or missing). The trend tests across quartiles of percent staining and the multiplicative score were conducted by assigning median values of these categories and entering these values as continuous terms in the Cox model.

To investigate whether the associations between PTEN expression and lethal prostate cancer risk differ between the HPFS and PHS cohorts, we conducted survival analyses in the HPFS and PHS separately and calculated the Q statistic, which follows an approximate χ² distribution with 1 degree of freedom. Because of the absence of heterogeneity, the 2 cohorts were merged for all analyses.

We assessed whether the association between PTEN expression and the development of lethal prostate cancer differed by IGF-IR expression in a subset of individuals (n = 651), who have both the PTEN and IGF-IR expression measured. IGF-IR expression was measured by a multiplicative score, similar to that of PTEN expression. The top tertile of the IGF-IR multiplicative score was arbitrarily defined as high IGF-IR expression. We conducted stratified survival analyses by the IGF-IR multiplicative score to evaluate the associations of percent PTEN staining and PTEN multiplicative staining score and lethal prostate cancer risk. We also generated interaction terms between PTEN expression measures and the IGF-IR multiplicative score and fitted multivariate Cox models with main effect measures and covariates. The significance of the interaction was determined using Wald test to test the β-coefficients of the cross-product terms.

We evaluated the ability of PTEN and IGF-IR expression to predict 10-year survival after diagnosis of prostate cancer using logistic regression, comparing the base model containing only clinical and demographic covariates to the model also containing PTEN and IGF-IR expression and their interaction. Area under curve (AUC), net reclassification improvement (NRI), and integrated discrimination index (IDI) were calculated for these 2 nested models.

All statistical analyses were 2-sided, and P < 0.05 was considered statistically significant. We conducted all analyses using the SAS software (SAS Institute, Inc., Version 9.1).

The baseline characteristics of the HPFS and PHS tumor cohorts are presented in Table 1. These 2 cohorts were demographically and clinically similar. In HPFS, 475 men who were diagnosed with prostate cancer between 1986 and 2002 have been followed prospectively for clinical progression and mortality, with a median follow-up of 13.2 years. The average age at cancer diagnosis was 65.6 years. A total of 38 prostate cancer–specific death and 10 cases of distant metastatic prostate cancer were documented. In PHS, 330 men were diagnosed with prostate cancer between 1983 and 2005. The mean age at diagnosis was 66.4 years. During a median follow-up of 10.9 years, 25 cancer deaths and 3 distant metastases occurred. For both cohorts, the majority of the cancers was diagnosed after the clinical introduction of PSA test as prostate cancer screening. The proportion of current smokers was relatively low at baseline in both cohorts, compared to the general population.

The characteristics of prostate cancer cases were compared across quartiles of PTEN expression, as shown in Table 2. No significant differences in age at cancer diagnosis, baseline BMI, or PSA at diagnosis were observed across quartiles of percent area for PTEN staining. Tumors with lower percent area for PTEN staining were more likely to be advanced stage (P_trend = 0.06) and higher Gleason grade (P_trend = 0.049). The results were similar when PTEN expression was stratified according to the multiplicative staining score (data not shown). Higher expression of PTEN appeared to be associated with more apoptosis in tumor cells (P_trend = 0.002). Such a trend was not present for either characteristics of angiogenesis including microvessel size and irregularity or cell proliferation.

We evaluated the association between PTEN expression in tumor and lethal prostate cancer (cancer death or distant metastasis); the results are shown in Table 3. Using the percent of tumor tissue with positive staining as a continuous variable, every 10% decrease in staining conferred a 20% increase in risk of lethal disease, after adjusting for age at diagnosis, diagnosis era, baseline BMI, and smoking status. Men with the lowest percent staining (bottom quartile) were at markedly higher risk for lethal cancer (HR, 2.4; 95% CI, 1.2–4.7), compared to those with the highest percent staining (top quartile; P_trend = 0.04). Low percent of tumor tissue stained positive for PTEN protein (<32%) was associated with a borderline significant HR of 1.7 (95% CI, 0.98–3.2). However, when we further adjusted for clinical characteristics of the tumor, such as tumor stage, Gleason grade, and PSA at diagnosis, percent of tumor tissue with PTEN expression no longer predicted lethal prostate cancer. The multiplicative score for PTEN staining, as a measure for total PTEN protein in tumor, yielded very similar findings.

To examine the potential interaction between PTEN and IGF-IR expression, we stratified the study population by IGF-IR staining score. As shown in Table 4, the associations between measures of PTEN expression and development of lethal prostate cancer varied according to the level of IGF-IR expression. When the expression of IGF-IR protein is low in the tumor, low PTEN expression was significantly predictive of lethal disease, adjusted for both demographic and clinical parameters. A statistically significant trend in risk of lethal prostate cancer was present across the quartiles of PTEN expression (P = 0.01 for percent and P = 0.04 for score). Low percent and low score for PTEN staining were both associated a >10-fold increase in the multivariate HR. However, when the expression of IGF-IR protein is high in the tumor, no association between measures of PTEN expression and lethal prostate cancer was observed. A strong negative interaction was observed between PTEN and IGF-IR expression (P = 0.03). In additional multivariate survival analyses presented in Fig. 1, we used men with low score for IGF-IR staining and high percent of tumor with PTEN staining as the reference group. High score for IGF-IR staining alone was associated with an HR of 13.8 (95% CI, 1.7–112.8). Similarly, low percent of tumor with PTEN staining alone was associated with an HR of 11.4 (95% CI, 1.5–84.3). However, combining these 2 conditions did not confer additional risk for lethal prostate cancer (HR, 12.0; 95% CI, 1.6–90.2).

To assess the ability of PTEN and IGF-IR expression to predict 10-year survival after diagnosis of prostate cancer, we compared the base logistic regression model that included demographic factors such as smoking status and BMI as well as prognostic factors including patient age, tumor stage, Gleason score, and PSA at diagnosis, to the model also including percent for PTEN staining, score for IGF-IR staining, and their interaction. Adding PTEN and IGF-IR expression to the base model increased AUC from 0.837 to 0.864 and improved NRI (NRI = 0.191, P = 0.004) and IDI (IDI = 0.0129, P = 0.0007) significantly. These results suggest that inclusion of PTEN and IGF-IR expression and their interaction in the base model offer further predicting power for prostate cancer prognosis.

To our knowledge, our study is one of the largest studies to evaluate the role of PTEN protein expression level in relation to the risk of lethal prostate cancer. We observed a significant positive association between reduction in PTEN protein expression and development of lethal disease, although the association was attenuated after adjustment for tumor stage, Gleason grade, and PSA levels. We are also the first to report that the association of PTEN tumor expression with clinical outcome is strongly modified by the level of IGF-IR expression in the tumor. These 2 factors negatively interact with each other: Unfavorable changes in protein expression of either biomarker alone are associated with increased risk, but combined unfavorable changes in both do not confer additional harm. PTEN and IGF-IR expression in tumor predicts prostate cancer survival independently of clinical prognostic factors.

Loss of PTEN expression has been shown to be correlated with indicators of poorer prognosis, such as high grade (17–20), advanced stage (18, 20), and the number of microvessels, a marker of angiogenesis (21). Several studies also reported that PTEN inactivation is associated with biochemical or clinical recurrence of prostate cancer (22–25). Three recent studies examined the effect of PTEN loss in relation to cause-specific death, which is the most relevant outcome for prostate cancer (18, 19, 26). Reid and colleagues conducted FISH assays in TMAs to detect PTEN loss in 308 patients with conservatively managed prostate cancer and reported that loss of PTEN alone was not associated with prostate cancer survival (18). Pourmand and colleagues used tissue microdissection and PCR/single-strand conformation polymorphism methods to analyze PTEN mutations in 51 prostate tumor specimens obtained from radical prostatectomy or transurethral resection (19). Patients with PTEN mutations were found to have lower survival rates (P = 0.001). When Cox regression models were used, with adjustment for Gleason grade, age, and PSA at diagnosis, PTEN mutations were no longer associated with death by prostate cancer (P = 0.60). In the study by Sircar and colleagues (26), TMAs were constructed from transurethral resection samples of 59 patients with hormone-refractory prostate cancer. The 4-color FISH strategy was used to evaluate genomic deletion of PTEN. The number of PTEN gene deletions (undeleted, heterozygous, and homozygous) was significantly correlated with prostate cancer–specific mortality (r = 0.332, P = 0.04). Yet presence versus absence of any PTEN deletion, by itself, was not correlated with cancer death (r = 0.189, P = 0.3). These findings also imply that the change in PTEN protein level is more relevant in association with prostate cancer survival.

The role of circulating IGF1 and its binding proteins such as IGFBP3 in prostate cancer has been extensively investigated (35–46). Evidence that high levels of circulating IGF1 significantly increase the risk of total prostate cancer became less than convincing with some recent reports of null findings (37, 41, 43). However, several studies, including the PHS and HPFS, have shown that IGF1 is more strongly associated with low-grade prostate cancer (35, 40, 45). A recent pooled analysis of individual patient data from 12 prospective studies yielded a significantly elevated risk for prostate cancer, comparing the highest versus the lowest quintiles of the serum IGF-1 concentration (46). This increased risk became more pronounced when the analysis was restricted to low-grade prostate tumors, suggesting poorly differentiated tumors, which are more autonomous, are less sensitive to the stimulus of IGF1. Such reduced dependence of IGF1 may be the result of alterations in expression of the downstream signaling pathway molecules, including those in the PI3K/Akt pathway. As described previously, loss of PTEN is strongly associated with high-grade prostate tumor. We also found that lower PTEN levels were significantly associated with higher Gleason grade. Thus, our finding that higher levels of IGF-IR expression in tumor was related to lethal prostate cancer when PTEN level was high whereas lower PTEN was related to lethal outcome regardless IGF-IR further supports the notion that poorly differentiated tumors are less sensitive to IGF-1/IGF-IR.

In addition to IGF-IR, IGF-I also binds to and activates the insulin receptor (IR) at a weaker affinity (29). However, no interaction between PTEN and IR expression was present in our study population (data not shown), suggesting the interaction between PTEN and IGF-IR is specific to the IGF1/PI3K/Akt signaling cascade and the involvement of insulin signaling may be limited.

There are potential limitations in our study to be considered. First, the PHS and HPFS Tumor Cohort consist of physicians and health professionals, are mainly Caucasian, and have relatively high socioeconomic status. Thus, it is not a random sample of the general population. This may limit the generalizability of the results, although the validity of the study is not undermined. We may be able to gain insights into the underlying biologic mechanism through which reduced PTEN expression may promote prostate cancer progression. Second, we have relatively few deaths or distant metastases from prostate cancer considering our large sample size. However, despite the small number of outcomes, we had sufficient power to detect the interaction between PTEN and IGF-IR expression. Third, the tumor specimens were obtained only when men underwent prostatectomy or TURP as curative treatment for their cancer. These are largely localized, organ-confined tumors. Those who were diagnosed with more advanced cancers may be ineligible for surgical intervention and have undergone hormonal, radiation therapy, or chemotherapy. As loss or reduction of PTEN is generally regarded as a late event in prostatic tumorigenesis and is associated with more malignant tumors and worse prognosis, it is likely that our analysis underestimates the role of PTEN expression in relation to prostate cancer mortality. The use of tumor blocks may generate certain heterogeneity for these tumors were diagnosed at different times and fixed and processed in different pathologic laboratories. Last, the presence of missing data such as PSA at diagnosis and baseline smoking status can lead to residual confounding by these factors. Uncontrolled confounding by unknown or unmeasured factors cannot be ruled out either.

The prospective study design with long-term and complete follow-up adds to the strength of our analysis. Clinical characteristics of the cancers were systematically extracted from medical records in a standardized format. The study pathologists reviewed the records and provided uniform Gleason grades for all cases. These approaches should contribute to reduction in measurement errors. In addition, we used cause-specific death and distant metastasis as the outcome, which are the most clinically relevant endpoints for prostate cancer.

In conclusion, low levels of PTEN protein are associated with an increased risk of lethal prostate cancer. This association is strongly modified by IGF-IR expression in the tumor. Our results reinforce the idea that the PI3K/PTEN/Akt pathway plays a critical role in prostate cancer survival. PTEN and IGF-IR are eligible independent predictors for prognosis. Their interaction may shine light on the complex picture of IGF axis in prostate tumorigenesis and cancer progression.

L.A. Mucci has a commercial research grant and is also a consultant/advisory board member of Metamark Genetics. No potential conflicts of interest were disclosed by the other authors.

Conception and design: K. Zu, N.E. Martin, M.N. Pollak, L.A. Mucci, E.L. Giovannucci

Development of methodology: K. Zu, R. Flavin, S. Finn, M.N. Pollak, L.A. Mucci, E.L. Giovannucci

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): N.E. Martin, R. Flavin, J. Ma, M.E. Gleave, M.J. Stampfer, M. Loda, L.A. Mucci, E.L. Giovannucci

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): K. Zu, M. Fiorentino, R.T. Lis, S. Finn, M.J. Stampfer, M.N. Pollak, M. Loda, E.L. Giovannucci

Writing, review, and/or revision of the manuscript: K. Zu, N.E. Martin, R. Flavin, R.T. Lis, J.A. Sinnott, K.L. Penney, J. Ma, M.J. Stampfer, M.N. Pollak, M. Loda, L.A. Mucci, E.L. Giovannucci

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): R.T. Lis, J. Ma, M.E. Gleave, M.N. Pollak

Study supervision: L.A. Mucci

Immunohistochemical evaluation and pathology: L. Fazli

Pathological analysis and evaluation: T.A. Bismar

The authors thank the participants and staff in the Physicians' Health Study for their ongoing participation and support and the participants and staff of the Health Professionals Follow-up Study, for their valuable contributions as well as the following state cancer registries for their help: AL, AZ, AR, CA, CO, CT, DE, FL, GA, ID, IL, IN, IA, KY, LA, ME, MD, MA, MI, NE, NH, NJ, NY, NC, ND, OH, OK, OR, PA, RI, SC, TN, TX, VA, WA, WY. They also thank Luba Bondarenko, Li Moy, and Julia Fleet for tumor block collection; Chungdak Li for construction of the tumor tissue microarrays; and Edward Stack for generating images on IHC staining of PTEN.

This project was funded by NIH R01 CA133891 (E.L. Giovannucci), CA136578 (L.A. Mucci), CA141298 (M.J. Stampfer), and US Army Prostate Cancer Program W81XWH-05-1-0562 (L.A. Mucci). The Physicians' Health Study is supported by grants CA-34933, CA-40360, and CA-091793 from the National Cancer Institute and grants HL-26490 and HL-34595 from the National Heart, Lung, and Blood Institute, Bethesda, MD. The Health Professionals Follow-up study is supported by NIH UM1 CA167552. N.E. Martin and L.A. Mucci are supported by the Prostate Cancer Foundation.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.