The mammalian target of rapamycin (mTOR) pathway is up-regulated in many human cancers, and agents targeting the mTOR pathway are in various stages of clinical development. The goal of the study was to evaluate the potential and limitations of targeting the mTOR pathway in renal cell carcinoma (RCC).
Immunohistochemical analysis using antibodies against pAkt, PTEN, p27, and pS6 was performed on a tissue microarray constructed from paraffin-embedded specimens from 375 patients treated by nephrectomy for RCC. The expression was associated with pathological parameters and survival.
The mTOR pathway was more significantly altered in clear-cell RCC, high-grade tumors, and tumors with poor prognostic features. PS6 and PTEN showed the strongest associations with pathological parameters. Survival tree analysis regarding expression of cytoplasmic pAkt, nuclear pAkt, PTEN, cytoplasmic p27, and pS6 identified staining percentages of 40%, 10%, 75%, 7%, and 70%, respectively, as ideal cutoff values for stratification, with corresponding P-values of .03, .001, .02, .005, and <.0001, respectively. Interestingly, high nuclear pAkt expression was associated with a favorable prognosis, whereas high cytoplasmic pAkt expression was associated with a poor prognosis. In multivariate Cox regression analysis, ECOG PS, T classification, N classification, M classification, cytoplasmic Akt, nuclear pAkt, PTEN, and pS6 were independent prognostic factors of DSS.
With over 38,000 new cases, representing an annual increase of 2% to 3%, and over 12,000 cancer-related deaths in 2006 in the US,1, 2 renal cell carcinoma (RCC) represents a major therapeutic challenge. Only a small number of patients with metastatic RCC can be cured by existing therapies. Approximately 20% to 30% of patients present with metastatic disease and an additional 20% to 40% develop recurrence after undergoing curative surgery for localized RCC.3, 4 Advances in biological and immune-based therapies have produced response rates for patients with metastatic RCC of approximately 15% to 30%, with some long-term durable remissions. Recent advances in understanding the changes associated with von Hippel-Lindau (VHL) gene inactivation have led to several angiogenesis inhibitors (sunitinib, sorafenib, bevacizumab) demonstrating enhanced response, improvement in progression-free survival, and trends toward improvement in overall survival for some of these agents administered in both the first- and second-line setting.5–11 A comparison of sunitinb to interferon-alpha (IFN) in a recent phase 3 trial demonstrating improvement in progression-free survival has changed the paradigm for treatment of this disease.11 Despite these advances, most responses are partial in nature, with the majority of patients ultimately succumbing to their disease.
Significant achievements in the basic sciences have led to a greater knowledge of the underlying signaling pathways in RCC,12 including the mammalian target of rapamycin (mTOR) pathway (phosphoinositide 3-kinase/Akt pathway) (Fig. 1). The mTOR pathway has a central role in the regulation of cell growth and increasing evidence suggests its dysregulation in cancer.13 Receiving input from multiple signals, including growth factors, hormones, nutrients, and other stimulants or mitogens, the pathway stimulates protein synthesis by phosphorylating key translation regulators such as ribosomal S6 kinase. The mTOR pathway also contributes to many other critical cellular functions, including protein degradation and angiogenesis. Hence, use of inhibitors of the pathway represents a new strategy for the targeted treatment of RCC.
Temsirolimus (CCI-779) is an inhibitor of mTOR and, in a phase 2 trial, showed antitumor activity in heavily pretreated patients with advanced RCC.14 In a recent randomized phase 3 trial of patients with poor prognosis, previously untreated, metastatic RCC, temsirolimus demonstrated a statistically significant 49% improvement in overall survival when compared with IFN.15
With mTOR inhibitors, it is unclear what clinical parameters and/or molecular pathways will predict which patients will derive the greatest benefit. These agents might have clinical activity only in selected patient cohorts in whose diseases this pathway drives their biology. An enhanced ability to predict patient survival would allow patients most likely to benefit from mTOR targeting therapies to be selected. For patient selection, a wide spectrum of molecular biomarkers is currently available including upstream and downstream targets of mTOR. Hence, the goals of our study were 1) to evaluate the prognostic relevance of the mTOR pathway in RCC in a large patient cohort, and 2) to identify patients whose tumor biology would most likely benefit from mTOR targeting therapy. For these goals, we carried out an immunohistochemical study of the mTOR upstream and downstream targets phosphorylated Akt (pAkt), phosphorylated S6 ribosomal protein (pS6), and p27, as well as the tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10), and correlated our findings with pathological parameters and survival.
MATERIALS AND METHODS
Our study cohort consisted of 375 patients who underwent radical or partial nephrectomy for sporadic RCC at the University of California, Los Angeles (UCLA) between 1989 and 2000. After approval by the UCLA Institutional Review Board, a retrospective study was performed with outcome assessment based on chart review of clinical and pathological data. Clinical data included age, gender, and Eastern Cooperative Oncology Group performance status (ECOG PS). Pathological data included TNM staging and histologic subtyping, which was performed according to the 1997 Union Internationale Contre le Cancer (UICC) and American Joint Committee on Cancer (AJCC) classification, as well as Fuhrman grade. Localized RCC was defined as N0M0 RCC, whereas metastatic RCC was defined if regional lymph node metastasis and/or distant metastasis were present.
Tissue Microarray Construction
Formalin-fixed, paraffin-embedded tumor specimens from our patient cohort were obtained from the Department of Pathology. Three core tissue biopsies, 0.6 mm in diameter, were taken from selected morphologically representative regions of each paraffin-embedded RCC and precisely arrayed using a custom-built instrument as described previously.16 Additional core tissue biopsies were taken from morphologically benign-appearing surrounding renal parenchyma tissue for each tumor. Sections of the resulting tumor tissue microarray block, 4 μm thick, were transferred to glass slides using the paraffin sectioning aid system (adhesive coated slides PSA-CS4x, adhesive tape, UV lamp, Instrumedics, Hackensack, NJ) to support the cohesion of 0.6 mm array elements.
Immunohistochemical Staining and Evaluation
Immunohistochemical staining was performed with a Dako Envision (Dako, Carpinteria, Calif) or Vectastain Elite ABC (Vector, Burlingame, Calif) staining system, as described previously.17, 18 Rabbit monoclonal antibody phospho-Akt Ser473 (Cell Signaling, Danvers, Mass) at a concentration of 1.5 μg/mL was used to stain for pAkt. Immunostaining for PTEN was performed using rabbit polyclonal antibody PN37 (Zymed, San Francisco, Calif) at 2 μg/mL. Mouse monoclonal antibody SX53G8 (Dako) was used at a concentration of 8 μg/mL to stain for p27. Staining for pS6 was performed with polyclonal rabbit antibody phospho-S6 ribosomal protein Ser 235/236 (Cell Signaling) at a concentration of 0.125 μg/mL.
The expression was evaluated by an anatomical pathologist (D.B.S.) in a blinded fashion to validate the diagnostic morphology of each array spot. The evaluation of expression involved site (subcellular localization) and degree of reactivity (staining intensity: 0 = negative, 1 = weak, 2 = moderate, 3 = strong, and staining frequency: percentage of positive cells). The overall score used for subsequent statistical analysis was the pooled mean from the 3 spots of the same tumor.
The associations between protein expression and T classification, Fuhrman grade, metastatic status, and histologic subtype were evaluated using the nonparametric Mann-Whitney U-test (when 2 independent groups were compared) or the Kruskal-Wallis test (when more than 2 independent groups were compared). Correlations were determined using the Pearson coefficient. The primary endpoint was disease-specific survival time (DSS). The Kaplan-Meier method was used to generate the survival functions. To find appropriate cutoffs for classifying patients according to the amount of expression, we used the recursive partitioning function in the R software (www.r-project.org).19 Subsequently, the dichotomized variable was used in univariate and multivariate survival analysis. Univariate survival analysis was performed by using the log-rank test and univariate Cox regression analysis. Independent prognostic variables of survival were identified with a multivariate Cox regression analysis. A significance level of .05 was used for all statistical tests. The statistical package SPSS (Chicago, Ill) was used for the analysis.
Our study cohort consisted of 375 patients, 252 men and 123 women with a median age of 61 years (range, 27–88 years). Characteristics of the patients are summarized in Table 1.
Table 1. Patient and Tumor Characteristics
ECOG PS indicates Eastern Cooperative Oncology Group performance status.
Expression and Association With Pathological Variables (Figs. 2, 3)
Because staining intensity and frequency showed high intercorrelation (cytoplasmic pAkt: R = 0.88, nuclear pAkt: R = 0.90, cytoplasmic p27: R = 0.84, nuclear p27: R = 0.91, PTEN: R = 0.61, pS6: R = 0.94, each P < .0001), we restricted subsequent analyses to staining frequency.
Anti-pAkt staining was seen in both cytoplasmic and nuclear cellular staining compartments. Cytoplasmic staining was detected in 93% of the RCCs. Higher cytoplasmic expression was noted in clear-cell than in nonclear-cell RCC, whereas no significant associations were found with other variables. Nuclear staining was positive in 61% of the tumors. Higher nuclear expression was observed in patients with localized disease. A significantly inverse correlation was found with tumor size (R = −0.13, P = .01).
The anti-PTEN antibody stained the tissues of the array in the cytoplasmic cellular compartment in 96% of the tumors. Tumors showed a lower expression than normal renal tissues. PTEN expression was higher in tumors with lower T classification (T1/2), nonclear-cell subtypes, and in localized RCC.
Cellular staining with anti-p27 antibody occurred in both nuclear and cytoplasmic compartments in 78% and 46% of the tumors, respectively. Nuclear expression was higher in clear-cell RCC and inversely correlated with tumor size (R = −0.12, P = .02). Cytoplasmic expression was higher in metastatic RCC.
Anti-pS6 staining was only seen in the cytoplasmic cellular compartment. Staining in tumors was noted in 85% of the RCCs, where it was generally increased compared with matched normal tissue. Significantly higher expressions were observed in tumors with higher T classification, higher Fuhrman grades, in metastatic disease, and clear-cell subtype.
Correlation Between Biomarkers
The mean expression of nuclear and cytoplasmic pAkt (R = 0.25, P < .001) and nuclear and cytoplasmic p27 (R = 0.14, P = .01) were significantly correlated with each other. In addition, cytoplasmic pAkt expression was significantly correlated with nuclear p27 (R = 0.32, P < .001), and pS6 (R = 0.18, P = .001). Nuclear pAkt expression was significantly correlated with nuclear p27 (R = 0.38, P < .001), PTEN (R = 0.13, P = .02), and pS6 (P = −.18, P = .001). PTEN expression was further correlated with nuclear p27 expression (R = 0.12, P = .04).
The median follow-up time was 56.9 months (range, 0.2–141.8) for the censored patients and 14.1 months (range, 0.4–115.0) for patients who died from RCC.
Performing survival tree analysis with regard to expression of cytoplasmic pAkt, nuclear pAkt, PTEN, cytoplasmic p27, and pS6 for all patients, we identified staining percentages of 40%, 10%, 75%, 7%, and 70%, respectively, as ideal cutoff values for further patient stratification. The corresponding P-values for the dichotomized patient cohort, calculated with the log-rank test, were .034, .001, .021, .005, and <.0001, respectively (Fig. 4). Notably, nuclear p27 expression was not associated with DSS. High nuclear pAkt expression was associated with favorable prognosis, whereas high cytoplasmic pAkt expression was associated with poor prognosis. In univariate Cox regression analysis, ECOG PS, T classification, N classification, M classification, Fuhrman grade, expression of cytoplasmic and nuclear pAkt, PTEN, cytoplasmic p27, and pS6 were all predictors of DSS. In multivariate Cox regression analysis, ECOG PS, T classification, N classification, M classification, Fuhrman grade, cytoplasmic and nuclear pAkt, PTEN, and pS6 were independent prognostic factors (Table 2).
Table 2. Multivariate Cox Regression Analysis for All Patients
We carried out further subanalysis dividing patients into localized and metastatic RCC at initial presentation. For patients with localized RCC (N0M0), pS6, nuclear p27, and nuclear pAkt expression provided additional prognostic information to ECOG PS, T classification, and Fuhrman grade (Fig. 5A). Using a cutoff value of 65%, lower staining of pS6 predicted a better prognosis than a higher staining (P = .0001). Higher nuclear p27 expression (cutoff 35%) also predicted a more favorable outcome (P = .039). Furthermore, higher nuclear pAkt expression (cutoff 5%) was associated with longer survival (P = .027). Expression of PTEN (cutoff 70%) also seemed to be associated with outcome, but this difference did not reach statistical significance (P = .079). Univariate Cox regression analysis showed prognostic significance for ECOG PS, T classification, Fuhrman grade, and expression of nuclear p27, nuclear pAkt, and pS6. In multivariate Cox regression analysis, ECOG PS, T classification, and expression of pS6 were independent prognostic factors of DSS (Table 3A).
Table 3. Multivariate Cox Regression Analysis for Patients (A) With Localized RCC and (B) With Metastatic RCC
In metastatic patients, expression of cytoplasmic pAkt, PTEN, and pS6 provided additional prognostic information (Fig. 5B). For pS6 (cutoff 70%), lower staining predicted a better survival than higher staining, median survival 20.2 months (±3.3, SE) vs 13.6 months (±1.8, SE), respectively (P = .002). In addition, higher staining of PTEN (cutoff 35%) predicted a median survival of 16.0 months (± 3.3, SE) vs 8.0 months (±1.0, SE) for lower PTEN expression (P = .041). Lower cytoplasmic pAkt expression (cutoff 35%) also predicted a more favorable prognosis, with a median survival of 25.5 months (±4.6, SE) vs 15.1 months (±1.5, SE) for higher expression (P = .045). In contrast, DSS of the metastatic patients was not significantly influenced by expression of p27 and nuclear pAkt. Univariate Cox regression analysis showed prognostic significance for ECOG PS, T classification, N classification, Fuhrman grade, expression of cytoplasmic pAkt, PTEN, and pS6. In multivariate Cox regression analysis, T classification, N classification, and pS6 were independent prognostic factors of DSS (Table 3B).
Herein, we report a tissue-microarray based immunohistochemical analysis of the mTOR pathway in RCC. We evaluated pAkt, PTEN, p27, and pS6 and associated the expression of each molecular marker with relevant pathological parameters and survival.
The ribosomal protein S6 reflects S6 kinase activity,20 and the phosphorylated S6 protein effects its downstream targets, altering mRNA translation.21 In our study, pS6 expression significantly increased with T classification and Fuhrman grade, and was significantly higher in metastatic patients and in clear-cell RCC. Further, pS6 was the strongest predictor of survival both in localized and metastatic RCC. This is of particular importance, because predicting which patients with localized disease will experience recurrence is difficult.22 Our recent studies indicate that p21 and p53 are able to identify groups who are at greater risk of recurrence.23, 24 In the current study, we identified pS6 and T classification as the strongest independent prognostic factors of DSS in localized RCC. Further, pS6 might be an important tool to stratify metastatic patients into different risk groups and to improve patient selection for mTOR targeted therapy.
Taking the findings on pS6 together, we hypothesize that patients with metastatic clear-cell RCC would benefit most from mTOR-targeted therapy because the pathway seems to be strongly activated in these patients. This hypothesis is further supported by a study of Cho et al.25 In that study, tissue specimens were obtained from 24 patients with metastatic RCC participating in a study with the mTOR inhibitor temsirolimus. Using a staining index that combines the frequency and intensity of staining, they demonstrated that high expression of S6 kinase was associated with response to temsirolimus. Further shown in a recent study on patients with recurrent glioblastoma multiforme, higher levels of phosphorylated p70S6 kinase in baseline tumor samples appeared to predict a patient population more likely to benefit from treatment with temsirolimus.26 However, objective response rates are not always predictive for patient survival.27 Additionally, pS6 can also be phosphorylated in a mitogen-activated protein kinase (MAPK)-dependent manner, presumably through p90 Rsk.28 This suggests a pathway for S6 phosphorylation that is independent of mTOR activation. In RCC with high activation of pS6 independent of the mTOR pathway, pS6 may then be less reliable as a surrogate marker for mTOR activation and response to mTOR inhibitors; however, the prognostic information provided by pS6 would be the same.
PTEN is a tumor suppressor protein that is encoded by the tumor suppressor gene PTEN. It has been shown that PTEN loss occurs during carcinogenesis and is associated with adverse prognosis in RCC.29, 30 In addition, Neshat et al.20 were able to demonstrate that PTEN-deficient tumors have an increased sensitivity to temsirolimus. Correlating to pathological parameters, we observed a significantly higher expression in RCC with lower T classification and localized disease. Furthermore, PTEN expression was significantly lower in clear-cell RCC. Taking these facts together, mTOR inhibitors would most likely benefit patients with metastatic clear-cell RCC and low PTEN expression.
The subcellular localization of p27 and pAkt is of importance for pathobiology. Many studies have shown that low nuclear staining of p27 is associated with poor prognosis.31–34 However, little knowledge exists regarding cytoplasmic mislocalization of p27. We were able to show that cytoplasmic mislocalization and consequently higher cytoplasmic expression is also a poor prognostic finding. This has been shown in other cancers,35, 36 but, to our knowledge, never before in RCC. The underlying mechanisms are poorly understood. It is speculated that pAkt induced phosphorylation mainly contributes to the cytoplasmic retention.37, 38 Because the growth-restraining activity of p27 depends on its nuclear localization, the aberrant localization may impair its tumor suppressor functions.39, 40 Thus, the mTOR pathway is increasingly activated in tumors with cytoplasmic mislocalized p27. In our study, cytoplasmic p27 expression was associated with metastatic RCC but not with histologic type. Therefore, we speculate that the cytoplasmic mislocalization of p27 occurs in metastatic patients irrespective of their histologic type.
Further, we could show that the subcellular localization of pAkt has a significant impact on pathology and survival. Akt, also called protein kinase B, regulates both growth and survival mechanisms by phosphorylating a wide spectrum of substrates in the cytoplasm and the nucleus. Mainly, Akt is stimulated by a second messenger generated from phosphoinositide 3-kinase (PI3K), phosphatidylinosital (3,4,5)- trisphosphate.41 Akt can mediate phosphorylation of mTOR,42–44 a process that occurs through inactivation of the tuberous sclerosis complex (TSC). The TSC complex is a heterodimer consisting of unphosphorylated TSC2 (tuberin) and TSC1 (hamartin). Akt, by phosphorylating TSC2, disrupts the TSC complex, allowing Rheb (Ras homolog enriched in brain) to bind ATP and convert from the inactive GDP state to the active GTP state. GTP-bound Rheb in turn activates mTOR.45, 46 Our study showed a higher pAkt expression in the cytoplasm than in the nucleus. Further, nuclear pAkt expression was significantly higher in localized than in metastatic RCC. Consequently, patients with localized RCC might have less activation of the mTOR pathway and therefore less aggressive tumors and better survival. Possibly, the recently described anti-oncogenic role of pAkt41 is more dependent on nuclear localization. In our study, cytoplasmic as well as nuclear pAkt expressions were independent prognostic factors of DSS. Further, when performing univariate analysis classifying the patients in localized and metastatic RCC, higher nuclear pAkt expression was associated with favorable prognosis in localized patients, whereas higher cytoplasmic pAkt expression was a poor prognostic finding in metastatic patients. In conclusion, the localization of pAkt seems to be highly relevant in both localized and metastatic RCC.
At present, there are at least 3 mTOR inhibitors that have been widely characterized in preclinical models and are in clinical development as anticancer agents: temsirolimus, AP23573, and RAD001 (Fig. 1), esters of rapamycin with improved bioavailability and formulation. Rapamycin and its derivatives first bind to FK506 binding protein 12 (FKBP12). The FKBP12/rapamycin complex then binds mTOR, inducing a G1 growth arrest rather than apoptosis. Completed clinical trials show safety and efficacy of mTOR targeting therapy in patients with RCC. In a randomized phase 2 trial of the mTOR inhibitor temsirolimus, Atkins et al.14 observed an objective response rate (complete and partial responses) of 7% in patients with advanced RCC and a clinical benefit rate (complete, partial, and minor responses and stable disease for at least 24 weeks) of 51%. As noted above, mTOR inhibitors induce G1 arrest rather than apoptosis, which may explain why rates of disease stabilization were higher than objective response rates. More recently, Hudes et al.15 presented the results of a randomized phase 3 trial of temsirolimus comparing temsirolimus alone with IFN alone and both combined. All enrolled patients were selected with poor prognostic factors. This trial demonstrated a statistically significant 49% improvement in median overall survival in advanced RCC patients treated with this mTOR inhibitor when compared with IFN in the first-line setting.
Some limitations of this study need to be acknowledged. The nature of this study was retrospective and therefore only limited conclusions can be drawn. The herein reported expressions, cutoffs, and their associations with survival have to be validated independently by other groups, if possible in a prospective setting. Also, it is reasonable to evaluate the proteins of the mTOR pathway in conjunction with other well-known prognostic relevant factors such as p21,24 p53,23 and CAIX47 to identify the marker with the greatest prognostic accuracy.
In conclusion, the mTOR pathway appears to be very important in patients with RCC. We were able to demonstrate highly significant associations with pathological parameters and, more important, with survival. Further, our analysis suggests that not all RCC tumor types are equally amenable to treatment strategies targeting the mTOR pathway, but that a majority of patients have at least 1 component of the mTOR pathway impacted. The results also suggest that the mTOR pathway is more significantly altered in clear-cell RCC, high-grade tumors, and tumors with poor prognostic features. Thus, these patients with a highly activated mTOR pathway may benefit most from therapy specifically targeting this pathway. However, patients with nonclear-cell tumors and highly activated pathway may also be candidates for targeted therapy. Of the different biomarkers, pS6 showed the highest associations with pathology and survival; however, it should be studied in prospective trials as a predictive marker for response and survival to mTOR targeted therapy.
Supported in part by Wyeth Research and The Richard and Nancy Bloch Kidney Cancer Research Fund.