2257
`
`Prognostic Relevance of the mTOR Pathway
`in Renal Cell Carcinoma
`ImplicationsforMolecularPatientSelectionforTargetedTherapy
`
`1
`Allan J. Pantuck, MD
`2
`David B. Seligson, MD
`1
`Tobias Klatte, MD
`2
`Hong Yu, MD, PhD
`1
`John T. Leppert, MD
`Laurence Moore, MD, PhD
`3
`Timothy O’Toole, PhD
`4
`Jay Gibbons, PhD
`Arie S. Belldegrun, MD
`1,5
`Robert A. Figlin, MD
`
`1
`
`3
`
`1 Department of Urology, David Geffen School of
`Medicine, University of California, Los Angeles,
`California.
`
`2 Department of Pathology and Laboratory Medi-
`cine, David Geffen School of Medicine, University
`of California, Los Angeles, California.
`
`3 Wyeth Research, Cambridge, Massachusetts.
`
`4 Wyeth Research, Pearl River, New York.
`
`5 Department of Medicine, David Geffen School
`of Medicine, University of California, Los Angeles,
`California.
`
`BACKGROUND. The mammalian target of rapamycin (mTOR) pathway is up-regu-
`lated 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).
`METHODS. 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.
`RESULTS. 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, cytoplas-
`
`mic 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 regres-
`
`sion analysis, ECOG PS, T classification, N classification, M classification, cyto-
`
`plasmic Akt, nuclear pAkt, PTEN, and pS6 were independent prognostic factors
`
`of DSS.
`CONCLUSIONS. Components of the mTOR pathway are significantly associated
`with pathological features and survival. Not all RCC tumor types seem to be
`
`equally amenable to mTOR targeted therapy. PTEN, pAkt, p27, and pS6 may
`
`serve as surrogate parameters for patient selection and predicting prognosis.
`
`Patients with a highly activated mTOR pathway should benefit most from this
`recommended. Cancer
`
`results
`our
`validation of
`therapy. External
`2007;109:2257–67. Ó 2007 American Cancer Society.
`
`is
`
`Supported in part by Wyeth Research and The
`Richard
`and Nancy
`Bloch
`Kidney
`Cancer
`Research Fund.
`
`The first and second authors contributed equally
`to this article.
`
`Address for reprints: Robert A. Figlin, MD, Divi-
`sion of Medical Oncology and Experimental Ther-
`apeutics, City of Hope National Medical Center
`and Beckman Research Institute, 1500 East
`Duarte Rd., Duarte, CA 91010; Fax:
`(310) 267-
`1491; E-mail: rfiglin@coh.org
`
`revision received
`Received January 9, 2007;
`February 5, 2007; accepted February 8, 2007.
`
`KEYWORDS: mTOR, S6, Akt, PTEN, p27, survival.
`
`W ith 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% de-
`velop 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 approxi-
`mately 15% to 30%, with some long-term durable remissions. Recent
`
`ª 2007 American Cancer Society
`DOI 10.1002/cncr.22677
`Published online 17 April 2007 in Wiley InterScience (www.interscience.wiley.com).
`
`

`

`2258
`
`CANCER June 1, 2007 / Volume 109 / Number 11
`
`advances in understanding the changes associated
`with von Hippel-Lindau (VHL) gene inactivation have
`led to several angiogenesis inhibitors (sunitinib, sora-
`fenib, bevacizumab) demonstrating enhanced re-
`sponse,
`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 sur-
`vival has changed the paradigm for treatment of this
`disease.11 Despite these advances, most responses are
`partial in nature, with the majority of patients ulti-
`mately succumbing to their disease.
`Significant achievements in the basic sciences
`have led to a greater knowledge of the underlying sig-
`naling pathways in RCC,12 including the mammalian
`target of rapamycin (mTOR) pathway (phosphoinosi-
`tide 3-kinase/Akt pathway) (Fig. 1). The mTOR path-
`way 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 cellu-
`lar
`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, tem-
`sirolimus 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 bio-
`markers 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
`
`FIGURE 1. PI3K-Akt-mTOR pathway. Activation of the pathway leads to
`phosphorylation of S6 kinase and 4E-BP, activating the former and inactivat-
`ing the latter. S6 kinase and 4E-BP are critical components of the general
`translation machinery. Cell cycle regulator p27 is also regulated through
`mTOR. The pathway is negatively regulated by PTEN and rapamycin analogs
`like temsirolimus.
`
`these goals, we carried out an immunohistochemical
`study of the mTOR upstream and downstream targets
`phosphorylated Akt (pAkt), phosphorylated S6 ribo-
`somal protein (pS6), and p27, as well as the tumor
`suppressor PTEN (phosphatase and tensin homolog
`deleted on chromosome 10), and correlated our find-
`ings with pathological parameters and survival.
`
`MATERIALS AND METHODS
`Patients
`Our study cohort consisted of 375 patients who
`underwent radical or partial nephrectomy for spo-
`radic 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.
`
`TABLE 1
`Patient and Tumor Characteristics
`
`mTOR Pathway in RCC/Pantuck et al.
`
`2259
`
`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 mor-
`phologically 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 tis-
`sue for each tumor. Sections of the resulting tumor tis-
`sue microarray block, 4 lm 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 anti-
`body phospho-Akt Ser473 (Cell Signaling, Danvers,
`Mass) at a concentration of 1.5 lg/mL was used to
`stain for pAkt. Immunostaining for PTEN was per-
`formed using rabbit polyclonal antibody PN37 (Zymed,
`San Francisco, Calif) at 2 lg/mL. Mouse monoclonal
`antibody SX53G8 (Dako) was used at a concentration
`of 8 lg/mL to stain for p27. Staining for pS6 was per-
`formed with polyclonal rabbit antibody phospho-S6
`ribosomal protein Ser 235/236 (Cell Signaling) at a
`concentration of 0.125 lg/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 lo-
`calization) and degree of reactivity (staining intensity:
`0 ¼ negative, 1 ¼ weak, 2 ¼ moderate, 3 ¼ 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.
`
`Statistical Analysis
`The associations between protein expression and T
`classification, Fuhrman grade, metastatic status, and
`histologic subtype were evaluated using the nonpara-
`metric Mann-Whitney U-test (when 2 independent
`groups were compared) or the Kruskal-Wallis test
`(when more than 2 independent groups were com-
`pared). Correlations were determined using the Pear-
`
`ECOG PS
`0
`1
`2
`X
`Tumor size
`Median
`Range
`T classification
`T1
`T2
`T3
`T4
`N classification
`N0
`N1
`N2
`M classification
`M0
`M1
`Fuhrman grade
`G1
`G2
`G3
`G4
`Histological subtype
`Clear cell
`Papillary
`Chromophobe
`Collecting duct
`
`No.
`
`149
`207
`16
`3
`
`6.5
`1–18
`
`140
`49
`165
`21
`
`323
`23
`29
`
`216
`159
`
`49
`187
`126
`13
`
`323
`40
`8
`4
`
`%
`
`39.7
`55.2
`4.3
`0.8
`
`—
`—
`
`37.3
`13.1
`44.0
`5.6
`
`86.1
`6.1
`7.7
`
`57.6
`42.4
`
`13.1
`49.9
`33.6
`3.5
`
`86.1
`10.7
`2.1
`1.1
`
`ECOG PS indicates Eastern Cooperative Oncology Group performance status.
`
`son 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 dichoto-
`mized variable was used in univariate and multivari-
`ate survival analysis. Univariate survival analysis was
`performed by using the log-rank test and univariate
`Cox regression analysis. Independent prognostic vari-
`ables of survival were identified with a multivariate
`Cox regression analysis. A significance level of .05
`was used for all statistical tests. The statistical pack-
`age SPSS (Chicago, Ill) was used for the analysis.
`
`RESULTS
`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.
`
`

`

`2260
`
`CANCER June 1, 2007 / Volume 109 / Number 11
`
`FIGURE 2. Immunohistochemical pattern of pAkt, PTEN, p27, and pS6 in (A) normal kidney tissue, (B) low-grade clear-cell renal cell carcinoma (RCC), and
`(C) high-grade clear-cell RCC.
`
`Expression and Association With Pathological
`Variables (Figs. 2, 3)
`Because staining intensity and frequency showed
`high intercorrelation (cytoplasmic pAkt: R ¼ 0.88,
`nuclear pAkt: R ¼ 0.90, cytoplasmic p27: R ¼ 0.84,
`nuclear p27: R ¼ 0.91, PTEN: R ¼ 0.61, pS6: R ¼ 0.94,
`each P < .0001), we restricted subsequent analyses to
`staining frequency.
`
`pAkt
`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 associa-
`tions were found with other variables. Nuclear stain-
`ing 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 ¼ 0.13, P ¼ .01).
`
`PTEN
`The anti-PTEN antibody stained the tissues of the
`array in the cytoplasmic cellular compartment in
`96% of the tumors. Tumors showed a lower expres-
`sion than normal renal tissues. PTEN expression was
`higher in tumors with lower T classification (T1/2),
`nonclear-cell subtypes, and in localized RCC.
`
`p27
`Cellular staining with anti-p27 antibody occurred in
`both nuclear and cytoplasmic compartments in 78%
`and 46% of the tumors, respectively. Nuclear expres-
`sion was higher
`in clear-cell RCC and inversely
`correlated with tumor size (R ¼ 0.12, P ¼ .02). Cyto-
`plasmic expression was higher in metastatic RCC.
`
`pS6
`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
`
`

`

`mTOR Pathway in RCC/Pantuck et al.
`
`2261
`
`FIGURE 3. Relation of T classification, Fuhrman grade, localized/metastatic disease, and histologic type and mean expression frequency. The Kruskal-Wallis
`test was used to compare expression among T classification and Fuhrman grade. Mann-Whitney U-test was applied to compare expression between localized
`and metastatic renal cell carcinoma (RCC), clear-cell vs nonclear-cell RCC, and T1/2 vs T3/4 (PTEN).
`
`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 ¼ 0.25, P < .001) and nuclear and cytoplas-
`mic p27 (R ¼ 0.14, P ¼ .01) were significantly corre-
`lated with each other. In addition, cytoplasmic pAkt
`expression was significantly correlated with nuclear
`p27 (R ¼ 0.32, P < .001), and pS6 (R ¼ 0.18,
`P ¼ .001). Nuclear pAkt expression was significantly
`correlated with nuclear p27 (R ¼ 0.38, P < .001),
`PTEN (R ¼ 0.13, P ¼ .02),
`(P ¼ .18,
`and pS6
`P ¼ .001). PTEN expression was further correlated
`with nuclear p27 expression (R ¼ 0.12, P ¼ .04).
`
`Survival Analysis
`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-
`.005, and <.0001,
`rank test, were .034,
`.001,
`.021,
`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 classi-
`fication, M classification, Fuhrman grade, expression
`of cytoplasmic and nuclear pAkt, PTEN, cytoplasmic
`p27, and pS6 were all predictors of DSS. In multivari-
`ate Cox regression analysis, ECOG PS, T classification,
`N classification, M classification, Fuhrman grade, cyto-
`plasmic and nuclear pAkt, PTEN, and pS6 were inde-
`pendent prognostic factors (Table 2).
`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 expres-
`sion 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 ¼ .0001). Higher nuclear p27 expression
`
`

`

`2262
`
`CANCER June 1, 2007 / Volume 109 / Number 11
`
`FIGURE 4. Kaplan-Meier survival estimates according to protein expression for all patients.
`
`(cutoff 35%) also predicted a more favorable out-
`come (P ¼ .039). Furthermore, higher nuclear pAkt
`expression (cutoff 5%) was associated with longer
`survival (P ¼ .027). Expression of PTEN (cutoff 70%)
`also seemed to be associated with outcome, but this
`difference did not
`reach statistical
`significance
`(P ¼ .079). Univariate Cox regression analysis showed
`prognostic significance for ECOG PS, T classification,
`Fuhrman grade, and expression of nuclear p27, nu-
`clear pAkt, and pS6. In multivariate Cox regression
`analysis, ECOG PS, T classification, and expression
`of pS6 were independent prognostic factors of DSS
`(Table 3A).
`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 stain-
`ing, median survival 20.2 months (3.3, SE) vs 13.6
`months (1.8, SE), respectively (P ¼ .002). In addi-
`tion, higher staining of PTEN (cutoff 35%) predicted
`a median survival of 16.0 months ( 3.3, SE) vs 8.0
`(1.0, SE)
`months
`for
`lower PTEN expression
`
`(P ¼ .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 ¼ .045). In
`contrast, DSS of the metastatic patients was not sig-
`nificantly influenced by expression of p27 and nu-
`clear pAkt. Univariate Cox
`regression analysis
`showed prognostic significance for ECOG PS, T clas-
`sification, N classification, Fuhrman grade, expres-
`sion of cytoplasmic pAkt, PTEN, and pS6.
`In
`multivariate Cox regression analysis, T classification,
`N classification, and pS6 were independent prognos-
`tic factors of DSS (Table 3B).
`
`DISCUSSION
`Herein, we report a tissue-microarray based immu-
`nohistochemical 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.
`
`

`

`TABLE 2
`Multivariate Cox Regression Analysis for All Patients
`
`Factor
`
`ECOG PS
`
`T classification
`
`N classification
`
`M classification
`
`Fuhrman grade
`
`p27 cytoplasmic
`
`pAkt cytoplasmic
`
`pAkt nuclear
`
`PTEN
`
`pS6
`
`Category
`
`Hazard ratio [95% CI]
`
`0
`1
`T1
`T2
`T3
`T4
`N0
`N 1
`M0
`M1
`G1
`G2
`G3
`G4
`<7%
`7%
`<40%
`40%
`<10%
`10%
`<75%
`75%
`<70%
`70%
`
`1
`2.14 [1.31, 3.49]
`1
`0.99 [0.49, 1.99]
`2.18 [1.28, 3.71]
`3.86 [1.70, 8.78]
`1
`2.14 [1.33, 3.42]
`1
`2.60 [1.67, 4.04]
`1
`3.65 [1.12, 11.94]
`4.08 [1.23, 13.55]
`2.84 [0.65, 12.45]
`1
`1.00 [0.69, 1.45]
`1
`1.69 [1.12, 2.54]
`1
`0.60 [0.41, 0.89]
`1
`0.67 [0.47, 0.95]
`1
`1.61 [1.11, 2.32]
`
`P
`
`.002
`
`.972
`.004
`.001
`
`.002
`
`<.001
`
`.032
`.022
`.167
`
`.997
`
`.012
`
`.011
`
`.024
`
`.011
`
`ECOG PS indicates Eastern Cooperative Oncology Group performance status; 95% CI, 95% confi-
`dence interval.
`
`The ribosomal protein S6 reflects S6 kinase activ-
`ity,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 signifi-
`cantly higher in metastatic patients and in clear-cell
`RCC. Further, pS6 was the strongest predictor of sur-
`vival both in localized and metastatic RCC. This is of
`particular
`importance, because predicting which
`patients with localized disease will experience recur-
`rence 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 strong-
`est independent prognostic factors of DSS in loca-
`lized 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 hypothe-
`size 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 sup-
`ported by a study of Cho et al.25 In that study, tissue
`
`mTOR Pathway in RCC/Pantuck et al.
`
`2263
`
`specimens were obtained from 24 patients with met-
`astatic 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. Fur-
`ther shown in a recent study on patients with re-
`current glioblastoma multiforme, higher levels of
`phosphorylated p70S6 kinase in baseline tumor sam-
`ples appeared to predict a patient population more
`likely to benefit from treatment with temsirolimus.26
`However, objective response rates are not always pre-
`dictive for patient survival.27 Additionally, pS6 can
`also be phosphorylated in a mitogen-activated pro-
`tein kinase (MAPK)-dependent manner, presumably
`through p90 Rsk.28 This suggests a pathway for S6
`phosphorylation that is independent of mTOR activa-
`tion. 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 prognos-
`tic 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 carcino-
`genesis 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 mislocaliza-
`tion 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
`retention.37,38 Because
`the
`growth-
`cytoplasmic
`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
`
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`CANCER June 1, 2007 / Volume 109 / Number 11
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`FIGURE 5. Kaplan-Meier survival estimates according to protein expression for patients (A) with localized renal cell carcinoma (RCC) and (B) with metastatic RCC.
`
`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 loca-
`lization of pAkt has a significant impact on pathology
`and survival. Akt, also called protein kinase B, regu-
`lates both growth and survival mechanisms by phos-
`phorylating a wide spectrum of substrates in the
`cytoplasm and the nucleus. Mainly, Akt is stimulated
`by a second messenger generated from phosphoino-
`sitide 3-kinase (PI3K), phosphatidylinosital (3,4,5)-
`trisphosphate.41 Akt can mediate phosphorylation of
`mTOR,42–44 a process that occurs through inactiva-
`tion of the tuberous sclerosis complex (TSC). The
`TSC complex is a heterodimer consisting of unphos-
`phorylated 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 acti-
`vates mTOR.45,46 Our study showed a higher pAkt
`
`expression in the cytoplasm than in the nucleus. Fur-
`ther, nuclear pAkt expression was significantly higher
`in localized than in metastatic RCC. Consequently,
`patients with localized RCC might have less activa-
`tion of the mTOR pathway and therefore less aggres-
`sive tumors and better survival. Possibly, the recently
`described anti-oncogenic role of pAkt41 is more de-
`pendent on nuclear localization. In our study, cyto-
`plasmic 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 nu-
`clear pAkt expression was associated with favorable
`prognosis in localized patients, whereas higher cyto-
`plasmic pAkt expression was a poor prognostic find-
`ing 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
`
`

`

`TABLE 3
`Multivariate Cox Regression Analysis for Patients (A) With Localized
`RCC and (B) With Metastatic RCC
`
`Category
`
`Hazard ratio [95% CI]
`
`P
`
`(A) With localized RCC factor
`ECOG PS
`
`T classification
`
`Fuhrman grade
`
`p27 nuclear
`
`pAkt nuclear
`
`pS6
`
`(B) With Metastatic RCC Factor
`ECOG PS
`
`T classification
`
`N classification
`
`Fuhrman grade
`
`pAkt cytoplasmic
`
`PTEN
`
`pS6
`
`0
`1
`T1-2
`T3-4
`G1-G2
`G3-G4
`<35%
`35%
`<5%
`5%
`<65%
` 65%
`
`0
`1
`T1-T2
`T3-T4
`N0
`N 1
`G1-G2
`G3-G4
`<35%
`35%
`<35%
`35%
`<70%
`70%
`
`1
`2.87[1.37, 6.00]
`1
`3.52 [1.72, 7.20]
`1
`1.69 [0.79, 3.58]
`1
`0.36 [0.12, 1.10]
`1
`0.66 [0.31, 1.38]
`1
`3.14 [1.50, 6.58
`
`1
`1.46 [0.80, 2.63]
`1
`1.82 [1.15, 2.90]
`1
`1.75 [1.15, 2.68]
`1
`1.14 [0.75, 1.74]
`1
`1.31 [0.83, 2.04]
`1
`0.74 [0.43, 1.28]
`1
`1.55 [1.02, 2.35]
`
`.005
`
`.001
`
`.175
`
`.072
`
`.268
`
`.002
`
`.215
`
`.011
`
`.010
`
`.532
`
`.245
`
`.285
`
`.042
`
`RCC indicates renal cell carcinoma; 95% CI, 95% confidence interval; ECOG PS, Eastern Cooperative
`Oncology Group performance status.
`
`and formulation. Rapamycin and its derivatives first
`bind to FK506 binding protein 12 (FKBP12). The
`FKBP12/rapamycin complex then binds mTOR, indu-
`cing a G1 growth arrest rather than apoptosis. Com-
`pleted clinical
`trials show safety and efficacy of
`mTOR targeting therapy in patients with RCC. In a
`randomized phase 2 trial of the mTOR inhibitor tem-
`sirolimus, 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
`
`mTOR Pathway in RCC/Pantuck et al.
`
`2265
`
`in advanced RCC patients treated
`overall survival
`with this mTOR inhibitor when compared with IFN
`in the first-line setting.
`this study need to be
`Some limitations of
`acknowledged. The nature of this study was retro-
`spective and therefore only limited conclusions can
`be drawn. The herein reported expressions, cutoffs,
`and their associations with survival have to be vali-
`dated 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.
`
`2.
`
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