Activated extracellular signal-regulated kinase is an independent prognostic factor in clinically confined renal cell carcinoma

Activated Extracellular Signal-Regulated Kinase Is an IndependentPrognostic Factor in ClinicallyConfined Renal Cell CarcinomaLee Campbell, PhD1; Rosa Nuttall, M Pharm1; David Griffiths, MB BCh2; and Mark Gumbleton, PhD1

BACKGROUND: Extracellular signal-regulated kinase (ERK) promotes proliferation, metastasis, and poor

survival in cancers of the breast, lung, and liver. Advanced localized renal cell carcinoma (RCC) is extraordi-

narily treatment resistant and has high recurrence rates despite surgery. Limited data exist regarding the

prognostic significance of activated (phosphorylated) ERK in RCC. The authors hypothesized that activated

ERK (pERK) promotes disease progression and metastasis in localized RCC and may be of value as a bio-

marker to predict disease recurrence. METHODS: The expression profile of pERK was examined by immu-

nocytochemistry using a tissue microarray constructed from 174 drug treatment–naive patients who had

undergone radical nephrectomy for localized RCC. Levels of tumor-cell specific pERK were scored and cor-

related with clinicopathologic parameters of RCC and disease-free survival. RESULTS: Immunostaining for

pERK was present in 36% of all RCCs, with a predominance found in the clear cell histologic subtype. High

expression was associated with increased tumor size, increased TNM stage, and vascular invasion. Patients

with pERK-positive tumors had a mean disease-free survival of 4.19 years, compared with 6.38 years for

patients with pERK-negative tumors (P < .001). Cox regression models revealed pERK to be a significant in-

dependent predictor of disease-free survival, with a hazards score of 2.9 (P < .001), a value similar to tumor

grade (hazards ratio, 3.01; P < .001). CONCLUSIONS: Expression of pERK is an independent prognostic fac-

tor in RCC that is associated with advanced and aggressive pathologic features of renal tumors and pre-

dicts the onset of metastasis in patients with localized disease. Cancer 2009;115:3457–67. VC 2009

American Cancer Society.

KEY WORDS: biomarkers, ERK-1/2, prognostic factors, renal cell carcinoma, tyrosine kinase inhibitors.

Renal cell carcinoma (RCC) is a heterogeneous group of highly vascularized invasive kidney tumors,comprising clear cell or conventional (accounting for 60%-80% of all RCC cases), papillary, chromo-phobe, and collecting duct subtypes.1 Currently, RCC represents 3% of all cancers and 2% of all cancer-related deaths and is also 1 of the few cancers whose incidence and mortality rates are growing steadily

Received: October 29, 2008; Revised: December 30, 2008; Accepted: January 5, 2009

Published online: June 12, 2009 VC 2009 American Cancer Society

DOI: 10.1002/cncr.24389, www.interscience.wiley.com

Corresponding author: David Griffiths, MB BCh, Department of Pathology, School of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, UK;

[email protected] or Mark Gumbleton PhD, Welsh School of Pharmacy, Cardiff University, King Edward VII Avenue, Cardiff CF10 3NB, UK;

[email protected]

1Welsh School Of Pharmacy, Cardiff University, Cardiff, United Kingdom; 2Department of Pathology, School of Medicine, Cardiff University, Cardiff,

United Kingdom

We thank Dr. Julia Gee, Mrs. Susan Kyme, and Mrs. Pauline Finley of the Tenovus Research Group (Cardiff University) for advice and assistance with

the immunocytochemistry procedures, and Chris von Ruhland for help with image acquisition.

Cancer August 1, 2009 3457

Original Article

worldwide.2,3 Approximately one-third of all RCCpatients will have established metastatic disease at firstpresentation; the remainder with clinically localizedtumors usually undergo curative radical nephrectomy.Approximately 35% to 45% of these will also eventuallydevelop disease recurrence with metastasis at distantsites.2,3 However, the clinical course of those presentingwith localized disease is difficult to predict, even withinpatients who have similar clinicopathologic parameters.This reflects intrinsic differences in the molecular and bio-logic profiles of individual tumors.4

Historically, survival rates for metastatic RCC rarely

exceed 5%, resulting from the inherently refractive nature

of tumors to standard chemotherapy, radiotherapy, and

hormonal therapy.5 The last decade, however, has wit-

nessed an explosion in the understanding of the biologic

behavior of renal neoplasms and the identification of how

key signaling cascades and their molecular determinants

integrate to drive disease progression. This preclinical evi-

dence has led to the emergence of rationally designed

novel drugs that specifically target these aberrant signaling

pathways.6-8 The most developed targeted therapies cur-

rently in use for the treatment of advanced localized and

metastatic RCC are sorafenib and sunitinib, 2 orally active

multikinase inhibitors, and temsirolimus, an inhibitor of

the mammalian target of rapamycin (mTOR).9 Indeed,

temsirolimus, when used as a single agent, is shown to sig-

nificantly improve overall survival of patients with meta-

static cancer compared with standard therapies for

RCC.10 Despite these advances, challenges within the

field still exist and include among others the need to 1)

optimize the use of such targeted therapies by identifying

patients with high-risk disease who are most likely to ben-

efit from their use as single agents or in combination, and

2) evaluate the efficacy of targeted therapies in nonclear

cell RCC subgroups for which no specific trials have been

reported.11 Therefore the utility of tumor biomarkers that

not only identify high-risk patients but also have value in

guiding treatment decisions for RCC is increasingly real-

ized.4,12-14

The epidermal growth factor receptor (EGF-R)/

Ras/extracellular signal-regulated kinase (ERK) axis is

shown to be mutationally activated or overexpressed in a

significant number (>30%) of all cancers.15 Once acti-

vated (phosphorylated), the terminal protein kinases,

ERK-1/2, are translocated to the nucleus, whereupon an

extensive array of transcription factors are activated,

including among others Elk-1, Fos, and Stat-3.16 These

regulatory mechanisms lead to global changes in gene

expression profiles that mainly, but not unequivocally,

result in promotion of cell growth. Aberrant ERK signal-

ing is associated with other cancer-related processes,

namely tumor angiogenesis, cell migration and invasion,

and drug resistance.17,18 Together this has led to intense

pharmaceutical interest in developing tyrosine kinase

inhibitors that target signal transduction cascades such

as the EGF-R/Ras/ERK and PI-3K/AKT/mTOR

cascades.19,20

Much of the current literature that lends support for

ERK-1/2 serving as a cancer driver has been generated by

the use of in vitro, ex vivo, and mouse cancer models, with

an actual paucity of studies that report its expression, ac-

tivity, and significance in clinical tumor specimens.21 Of

the clinical immunohistologic studies undertaken to date,

phosphorylated ERK (pERK) has been shown to correlate

with disease progression in melanoma,22 hepatocellular,23

and nonsmall cell lung carcinomas.24 In hepatocellular

carcinoma, pERK is predominantly expressed in poorly

differentiated tumors, and as such serves as a powerful

prognostic factor.23 In this latter study, pERK also was

found to be correlated with the presence of intrahepatic

metastasis, and could retrospectively predict disease recur-

rence in patients after the complete surgical removal of

initial tumors.23 Trends of increasing levels of pERK have

also been observed in late-stage disease in both lung carci-

noma23 and melanoma,22 but it is not an independent

prognostic factor in either disease when analyzed for

patient survival. Conversely, the expression of pERK-1/2

in endometrial cancer25 has been shown to be an inde-

pendent prognostic indicator associated with a favorable

prognosis. Specifically, patients whose tumors exhibited

low levels of pERK-1/2 actually had significantly lower

disease-free survival and worse overall survival.25 A di-

chotomy in the impact of activated ERK also exists within

the same cancer type. For example, in some breast cancer

patient cohorts, high activated ERK-1/2 levels are report-

edly associated with early stage tumors and negative

lymph node status, and predict long disease-free sur-

vival.21 In others, its high expression is reported to corre-

late with advanced localized or metastatic disease,

tamoxifen resistance, and reduced time to disease recur-

rence.26 These apparent contradictions may reflect the

Original Article

3458 Cancer August 1, 2009

disease stage and context dependency of ERK-1/2. In sup-

port of this notion, in vitro studies have shown that varia-

tions in the magnitude and spatiotemporal expression of

pERK-1/2 can either potentiate proliferation or alter-

nately drive cellular differentiation and senescence.27

The prognostic significance of activated ERK-1/2 in

RCC remains undetermined. To test the hypothesis that

constitutively activated ERK-1/2 may potentiate disease

progression in RCC, we have examined, using tissue

microarray technology, the expression of pERK-1/2 in

176 RCCs resected from patients who had undergone

radical nephrectomy for the treatment of localized disease.

The relative expression of activated ERK-1/2 was corre-

lated with clinical and pathologic variables of RCC, with

the overall aim of determining whether pERK-1/2 might

have prognostic value in RCC patients.

MATERIALS AND METHODS

Patient Selection

Archival tumor specimens were identified from the patho-

logic records and consisted of a consecutive series of 174

patients who had undergone curative radical nephrectomy

for primary kidney tumors presenting between 1992 and

1996. This series of patients (for which ethical approval

was granted by the National Health Service local research

ethics committee) has been previously described else-

where.28 Tissue blocks, histology reports, and slides were

available in all cases without a priori knowledge of clinical

outcome. For each renal carcinoma, a tissue block was

selected that contained a sample of peripheral tumor for

making the tissue microarray (TMA). Tumors were classi-

fied according to: the Hiedelberg system29 and Furhman

nuclear grade30; the presence or absence of any vascular

invasion (either microvascular invasion, renal vein inva-

sion, or inferior vena cava invasion)31; and whether or not

there was capsular invasion with cellular invasion of peri-

nephric fat.32 No patients had received drug treatment or

had evidence of lymph node or distant metastatic disease

before or at surgery.

The median age of the 174 patients was 65 years

(range, 34 years-88 years); 119 were men and 55 were

women. Complete clinical follow-up was performed as

previously described.31,33 Briefly, patients had been

reviewed annually as out-patients for 3 to 7 years (mean

follow-up, 4.3 years; median follow-up, 3.92 years [range,

0.3 years-8.09 years]) with the following information

extracted from their records: date of birth, sex, date of sur-

gery, date patient last seen, date on which recurrent or

metastatic disease was first identified, and date and cause

of death. For patients in whom the cause of death was

recorded as RCC but whose time of recurrence was

unavailable, the date of death was considered the endpoint

for disease-free survival.

TMA Construction and

Immunocytochemistry

The customized TMA was constructed with a Beechers

manual tissue arrayer (Beechers Instruments, Inc, Sun

Prairie, Wis) from archived paraffin-embedded renal tu-

mor samples. A single core of representative peripheral tu-

mor (0.6 mm in diameter) was punched from each donor

block, using a specific orientation transplanted into a pre-

molded recipient paraffin wax block. Peripheral tumor

refers to representative viable tumor tissue taken within 5

mm of the growing edge of the tumor. Additional cores

were taken from normal renal tissue (adjacent to some of

the tumors). Serial sections were cut from the resulting

TMA block onto cleaned adhesive glass slides (Superfrost

Plus, Microm International, Walldorf, Germany) at 4 lmthickness.

Array sections were dewaxed using a sequential series

of graded xylenes and alcohols. The detection of pERK-

1/2 was undertaken using previously published proce-

dures.26 Briefly, after removal of paraffin wax, the endoge-

nous peroxidase activity within the rehydrated tissue was

quenched (3% hydrogen peroxide for 5 minutes). Antigen

retrieval consisted of microwaving TMA sections in citric

acid (0.1 M, pH 6.0) for 30 minutes, followed by slide

cooling with running tap water. After draining, the sec-

tions were equilibrated in 20% normal human serum

(Golden West Biologicals Inc, Temecula, Calif) for 15

minutes at room temperature (diluent, phosphate-buf-

fered saline [PBS]). The primary rabbit antihuman

pERK-1/2 antibody (New England Biolabs, Hitchin,

UK) was applied to each section at a dilution of 1:25 and

incubated overnight at 4�C for a total of 16 hours. There-

after, the sections were washed (4 � 1 minutes) with PBS

(pH 7.3), and tissue was immunostained using the

DAKO rabbit Envision staining system (DAKO,

Prognostic Significance of pERK in RCC/Campbell et al


Cambridge, UK) according to the manufacturer’s instruc-

tions. The TMA sections were counterstained with hema-

toxylin and finally mounted.

Controls and Scoring of Stained Specimens

Human breast cancer specimens, known to be positive for

pERK-1/2, were run in parallel and served as an appropri-

ate control for evaluation of staining in the kidney tissue.

Negative controls consisted of RCC TMA and breast can-

cer sections in which the primary antibody had been omit-

ted and replaced with 20% normal human serum

(isotypic control).

Scoring of tumor arrays was performed by a patholo-

gist (D.G.) and research associates (L.C. and R.N.) with-

out knowledge of other pathologic and clinical data.

Expression of pERK-1/2 was assessed semiquantitatively

accordingly to previously described criteria,28,33 with

slight modifications. This scoring system accounts for

both the intensity of immunostain within the nucleus and

cytoplasm of tumor cells and the percentage of tumor cells

involved in each core. Scoring was as follows: 0 indicates

no detectable reaction product (deposit) in tumor cells; 1

indicates very light diffuse or focal light deposit in tumor

cells; 2 indicates light diffuse or moderate focal deposit

(may include very small areas of heavy deposit); and 3

indicates tumor cores containing areas of heavy deposit in

most or all tumor cells.

Data and Statistical Analysis

Analysis of disease-free survival of patients with tumors

demonstrating different scores of staining for pERK-1/2

was performed using the Kaplan-Meier method using

the log-rank test, in which the first appearance of metas-

tasis was considered an event. Patients last seen alive

without metastasis or who died because of causes other

than RCC were considered censored at the date of last

contact or death, respectively. Scores were also con-

verted to a binary simple covariate designated positive

or negative by thresholding according to the most in-

formative split on the initial Kaplan-Meier analysis. For

pERK-1/2, a score of 0 was negative, and a score of 1 or

2 or 3 was positive. The association of positive pERK-

1/2 expression with recognized histologic tumor prog-

nostic variables (grade, size, vascular invasion, capsular

invasion, and tumor type) was examined by cross-tabu-

lation and the chi-square test.

Multivariate analysis by Cox regression was per-

formed to determine whether positive pERK-1/2 expres-

sion had influence on prediction of disease outcome in

relation to other clinical prognosticators for RCC. Previ-

ous analysis had already determined that the most influen-

tial covariates predicting disease-free survival of these

patients are Fuhrman grade (grades 1 and 2 and grades 3

and 4, respectively, are pooled for analysis), any degree of

vascular invasion (histological correlate of stage T2b), and

invasion of perinephric tissues (ie, capsular invasion and

histological correlate of stage T2a). When these covariates

are taken into consideration, tumor size and type were

found to have no influence on disease-free survival. There-

fore, in this current computation, pERK-1/2 was entered

into the multivariate analysis as an independent covariate,

using the enter function together with the covariates

grade, presence or absence of vascular invasion, and inva-

sion of perinephric tissue.

RESULTS

Immunohistochemistry for Activated ERK-

1/2 in Clinically Confined RCC

Of the 174 cases, 164 were assessable on the array; of

these, 60 (36%) stained positive for pERK-1/2. Immuno-

reactivity for pERK was detected in both the nucleus and

cytoplasm of tumor cells. In all cases examined, some

degree of cytoplasmic staining coexisted with nuclear

staining (Fig. 1). Cases that scored 1, in which staining

was predominantly light and diffuse, comprised 50% (30

of 60) of all pERK-positive cases. Moderate staining

(score 2) and more extensive heavy immunoreactivity

(score 3) accounted for 35% (21 of 60) and 15% (9 of 60)

of cases, respectively (Fig. 1).

Cross-tabulation of pERK-1/2 with conventional

determinants for RCC is shown in Table 1. Increased

expression levels of pERK-1/2 was found to be correlated

with tumor size (P ¼ .039), TNM staging (P ¼ .013),

and vascular invasion (P ¼ .022). No correlation was

observed between tumor grade and capsular invasion.

Notably increased expression of pERK-1/2 was found to

be significantly associated (P ¼ .006) with nonpapillary

tumors (of which 98% were of the clear cell type) when

Original Article


FIGURE 1. This is a representative tissue microarray of clinically confined renal cell carcinoma demonstrating the immunohisto-

chemical expression intensity and pattern of phosphorylated extracellular signal-regulated kinase in tumors that were typically

(A) score 0 (negative), (B) score 1, (C) score 2, and (D) score 3.



compared with the papillary subtype. It is interesting to

note that only 25 of 104 (24%) of patients with pERK-

negative tumors developed disease recurrence, compared

with 40% (12 of 30), 57% (12 of 21), and 56% (5 of 9) of

patients whose tumors had pERK positive scores of 1, 2,

and 3, respectively; note that pERK positive-tumors

accounted for 53% (29 of 54) of the total number of

recurrences within this patient cohort (Table 1).

Activated ERK-1/2 and Disease-free

Survival Analysis in Clinically Confined RCC

Univariate survival analysis (Kaplan-Meier) indicated that

patients with tumors that did not express pERK-1/2 had a

mean disease-free survival of 6.38 years (95% confidence

interval [95% CI], 5.80 years-6.96 years) compared with

4.83 years (95% CI, 3.68 years-5.98 years) for patient

samples scored as 1, 3.53 years (95% CI, 2.12 years-4.95

years) for patient samples scored as 2, and 3.93 years

(95% CI, 2.15 years-5.70 years) for patient samples

scored as 3. Therefore, increasing levels of pERK-1/2 in

tumor samples resected from RCC patients resulted in a

significantly (P ¼ .0017) shorter time to disease recur-

rence (Fig. 2 Top). This survival analysis also indicated

that patients whose tumors displayed any evidence of

pERK-1/2 staining had significantly shorter time to dis-

ease recurrence than patients whose tumors were negative

(Fig. 2 Top). This allowed a separate analysis to be under-

taken, in which tumors simply could be categorized as

positive or negative. In this analysis, patients with pERK-

1/2–positive tumors had a highly significant (P ¼ .0007)

reduced disease-free survival of 4.19 years (95% CI, 3.36

years-5.03 years), compared with 6.38 years (95% CI,

5.80 years-6.96 years) for patients with pERK-1/2–nega-

tive tumors (Fig. 2 Bottom). By implication, expression of

pERK-1/2 per se can be used to predict disease-free sur-

vival in patients with RCC.

Cox Regression Multivariate Analysis

By using multivariate Cox proportional hazards regression

models, we next evaluated whether the expression of

pERK-1/2 could be of prognostic value in the assessment

of primary renal tumors (Table 2). We have previously

shown31 the covariates of tumor grade, vascular invasion,

and capsular invasion to be influential prognostic factors

in RCC, but when tumor size and tumor type were not.

Therefore, tumor grade, vascular invasion, and capsular

invasion together with pERK were entered into the

model. The analysis revealed that pERK-1/2 was a

Table 1. Relation Between pERK-1/2 Expression Score and Conventional Clinicopathologic Parameters in Patients WithClinically Confined RCC*

Prognostic Indices Phosphorylated ERK-1/2† Recurrence,n (%)

0, n5104 1, n530 2, n521 3, n59 Total, n5164

Tumor grades 1 and 2 75 23 12 8 118 25 (21)

Tumor grades 3 and 4 29 7 9 1 46 29 (63)

TNM T1 50 13 3 1 67 10 (15)

TNM T2 28 5 8 2 43 10 (23)

TNM T3 and T4z 26 12 10 6 54 34 (63)

Tumor size <7 cm 55 17 4 3 79 17 (22)

Tumor size >7 cmz 49 13 17 6 85 37 (44)

Vascular invasion absent 71 15 11 4 101 18 (18)

Vascular invasion presentz 33 15 10 5 63 36 (57)

Capsular invasion absent 90 26 15 6 137 32 (23)

Capsular invasion present 14 4 6 3 27 22 (82)

Nonpapillaryz 84 29 21 8 142 51 (36)

Papillary 20 1 0 1 22 3 (14)

Recurrence, n (%) 25 (24) 12 (40) 12 (57) 5 (56) 54 (36%)

pERK indicates phosphorylated extracellular signal-regulated kinase; RCC, renal cell carcinoma.

* For information purposes, the percentage of disease recurrence in each category is included.

yFor contingency tables of the individual prognostic and pERK-1/2 covariates, the P values derived from chi-square testing are as follows: grade, P ¼ .755;

TNM T classification, P ¼ .013; tumor size, P ¼ .039; vascular invasion, P ¼ .022; capsular invasion, P ¼ .083; and nonpapillary versus papillary, P ¼ .006.

zDenotes statistical significance (P < .05) with respect to pERK-1/2.

Original Article


significant influential predictor of shortened disease-free

survival with a hazards ratio (HR) of 2.96 (P < .001). Of

note, the predictive value of pERK-1/2 was significantly

higher than the HR of vascular invasion (HR, 1.58; P ¼.20) and only slightly lower than that of the powerful

and robust prognostic indicator tumor grade (HR, 3.01;

P< .001).

DISCUSSION

Previous clinicopathologic studies have shown that acti-

vated ERK is a reliable predictor of disease progression in

several diverse cancers that include malignancies of the

liver,23 lung,24 and breast.26 Oka et al34 demonstrated a

statistically significant association between pERK expres-

sion and grade in a small series of 25 patients with RCC.

However, they determined activated ERK levels through

Western blot analysis of whole tumor homogenates, an

approach that suffers from population averaging effects

of contaminating nontumor cells contributed by stromal

and vascular compartments. Although experimental

studies with renal cancer cell lines and tumor xenografts

have provided important insights into the role of pERK

in several disease processes, such as invasion and angio-

genesis,35-38 the significance of pERK with respect to

disease progression and survival of patients with RCC

remains unknown. In this current study, we used immu-

nocytochemistry and TMA technologies to examine the

prognostic relevance of pERK expression in 164 cases of

RCC. To allow the direct assessment of the role of

pERK expression in disease progression and to circum-

vent the issues of drug treatment and coexistent meta-

static disease in data interpretation, our unique patient

cohort was selected on the basis that subjects: 1) pre-

sented with clinically confined disease only, and 2) had

not received any therapy before surgery. Herein we pro-

vide what to our knowledge is the first demonstration

that pERK expression correlates with aggressive and

advanced histopathologic indices of RCC and poor dis-

ease-free survival. In addition, our analysis also demon-

strated that pERK serves as a highly significant and

independent prognostic biomarker that predicts disease

progression in RCC.

In this study, increased expression of pERK-1/2 was

found to be correlated with the conventional

Table 2. Multivariate Cox Regression Hazards Model forTime to Disease Recurrence

Prognostic Indice/ModelIncluding VascularInvasion (No.)

HR 95% CI P-value

Grade 1 and 2 (118) 1

Grade 3 and 4 (46) 3.01 1.59-5.72 <.001

No capsular invasion present (137) 1

Capsular invasion present (27) 4.04 2.12-7.69 <.001

No vascular invasion present (101) 1

Vascular invasion present (63) 1.58 0.78-3.19 .20

pERK-1/2 negative (104) 1

pERK-1/2 positive (60) 2.96 1.69-5.67 <.001

HR indicates hazards ratio; 95% CI, 95% confidence interval; pERK, phos-

phorylated extracellular signal-regulated kinase.

FIGURE 2. Kaplan-Meier metastasis-free survival plots of re-

nal cell carcinoma patients with clinically confined disease

are (Top) based on phosphorylated extracellular signal-regu-

lated kinase (pERK) score and (Bottom) stratified by ‘‘posi-

tive’’ versus ‘‘negative’’ pERK expression.



clinicopathologic variables of tumor size and vascular

invasion. The association of pERK-1/2 with tumor size is

perhaps expected, given that ERK activation has been his-

torically connected with cell proliferation. Nevertheless,

this correlation is highly relevant, because tumor size is an

important prognosticator for patient survival in RCC,39

and is a histopathologic criterion that is incorporated into

the universally accepted TNM classification system used

for staging renal tumors.40 In addition to its direct effect

on cancer cell proliferation, pERK-1/2 may also contribute

to tumor size in RCC via the promotion of angiogenesis,

thereby maintaining the supply of oxygen and nutrients to

the tumor, affording uncompromised growth. Hypoxia-in-

ducible factor 1a (HIF-1a), a key molecule that facilitates

the process of angiogenesis in RCC, is a well-documented

substrate of ERK in several different cell types.41,42 The

cooperativity of HIF-1a and pERK-1/2 may explain the

high vascular nature of RCC tumors per se and hence the

correlation between pERK-1/2 and tumor size observed in

our current study. Consistent with this view, a recent study

has shown that the specific pharmacologic inhibition of

ERK is sufficient to suppress the growth and angiogenesis

of RCC tumor human xenografts in mice.37

During the final drafting of our current article, a

report by Lee et al43 was published that examined the

prognostic significance of pERK-1/2 in 328 patients with

RCC, comprising patients with localized and metastatic

clear-cell subtypes. These authors stratified patients into

low and high pERK-1/2 groups, and reported that

although pERK-1/2 expression did not reach any inde-

pendent prognostic significance, high pERK-1/2 expres-

sion as compared with low expression was correlated with

better disease-free survival, but only in patients whose

tumors measured<7 cm in greatest dimension; in tumors

measuring >7 cm, they did not demonstrate any correla-

tion between pERK-1/2 and survival. Increased tumor

size appears to be positively correlated with RCC progres-

sion. In our own study, restricted to localized RCC and

stratifying patients into positive and negative pERK-1/2

groups, we were able to demonstrate that pERK-1/2 was

an independent poor prognostic factor irrespective of tu-

mor size, although we did note a statistically higher inci-

dence of pERK-1/2 in tumors measuring >7 cm. In our

study, papillary tumors represented 13.4% of total RCCs

(22 of 164), which is typical of any given RCC patient

cohort. Analysis of the nonpapillary cohort still demon-

strated that increased pERK-1/2 levels served as a signifi-

cant predictor of disease recurrence in patients with

clinically confined RCC (P ¼ .018). Ethnic differences

between the patient cohort used in our study and that of

Lee et al cannot be dismissed as a basis for contrasting

findings. Ethnicity can provide for different genetic and

epigenetic backgrounds in common tumors that ulti-

mately impact on disease progression and response to

treatment. For example, EGF-R, a key upstream effector

molecule of ERK, demonstrates more frequent mutations

in Asian nonsmall cell lung carcinoma populations.44

Although immunohistochemistry provides opportunity

to determine both the global and cellular distribution of

pERK-1/2 within renal tumors, it does not allow discrimi-

nation between the exact involvement of the individual

activated ERK isotypes (ie, pERK-1 vs pERK-2) in disease

progression. The possibility exists that the mouse mono-

clonal antibody used in the study by Lee et al43 and the

rabbit polyclonal antibody used by ourselves in this cur-

rent study preferentially detect different isoforms of

pERK. Of note, gene ablation studies in mice and in vitro

cell lines have recently shown that ERK-2 predominately

drives Ras-dependent cell proliferation,45-47 whereas

ERK-1 can actually attenuate Ras-dependent tumor for-

mation.47 Using Affymetrix microarray technology,

Huang et al37 recently demonstrated elevated ERK-2 lev-

els in 174 cases of clear cell renal tumors.

Multiple reports have documented the importance

of microvascular tumor invasion as a robust and accurate

determinant of disease progression in RCC. Indeed, in

patients with low-grade and localized disease, vascular

invasion has been shown to be the most significant inde-

pendent prognosticator of all pathologic parameters

examined. The current study finding that pERK expres-

sion is strongly associated with the presence of vascular

invasion in clinically confined disease is highly relevant

and supports the inclusion of pERK status in staging

schemes and prognostic models for RCC that incorporate

tumor biomarkers alongside conventional parameters.

The clinical correlation between pERK expression and

invasion can be reconciled at the molecular level through

activated ERK induction of extracellular matrix-digesting

enzymes, facilitating cell migration, and invasion into sur-

rounding tissue.48-51 Recently, it has been demonstrated

that the invasive capacity of the human renal cell carci-

noma cell line Caki-2 is substantially increased on

Original Article


sustained activation of ERK-1/2.38 The enhanced ERK-

mediated invasive ability of these cells resulting from up-

regulation of metalloproteinases 2 and 9, a mechanism

attenuated by the combination of small interfering RNA–

mediated down-regulation of ERK-1/2 and chemical in-

hibition of the RAS/RAF/ERK pathway using PD98059.

Furthermore, it has been reported that leptin36 can poten-

tiate the invasiveness of murine renal cancer cells in an

ERK-dependent manner. Collectively, such mechanistic

studies support the view that activated ERK has a direct

involvement in the process of vascular invasion and hence

the promotion of metastatic disease in RCC, a view fur-

ther corroborated by our current clinical findings.

In the current study, multivariate Cox regression

analysis determined that pERK was equivalent to tumor

grade with respect to its ability to predict disease recur-

rence, with calculated hazards scores of 2.96 and 3.01,

respectively. Surprisingly, we failed to demonstrate a cor-

relation or trend between pERK expression and tumor

grade. However, because they are independent of each

other and yet represent highly valid independent prognos-

ticators, both pERK and tumor grade may be viewed as

complementary determinants that could be effectively

used for the improved prediction of metastatic disease in

clinically confined RCC. Prognostic models incorporat-

ing individual biomarkers with standard clinical parame-

ters have been developed for RCC with good effect.52,53

For example, malignancy-associated biomarkers that

include, among others, PTEN, p53, and Ki-67 have been

ranked alongside several conventional RCC staging

schemes such as Fuhrman grade, TNM, and Eastern Co-

operative Oncology Group performance status. The re-

sultant integrated ‘‘clinical/biomarker’’ allows better

prediction of patient survival than either the panel of bio-

markers or clinicopathologic parameters alone.

With the emergence of cancer therapies that specifi-

cally target growth factor signaling pathways, attention

has focused on the immunohistochemical evaluation of

suitable biomarkers that can serve as a basis for patient

selection and also as markers of treatment response. ERK

represents a downstream convergence point for several

disparate growth factor signaling cascades, including

among others EGF-R, platelet-derived growth factor re-

ceptor (PDGF-R), and vascular endothelial growth factor

receptor (VEGF-R).16-18 In our study, we demonstrated

that pERK-1/2 positivity per se is sufficient to signifi-

cantly predict disease progression in RCC without the

need to discern between various degrees of expression and

ascertain its exact subcellular localization. However,

pERK-1/2 may also represent a reliable and robust bio-

marker for the selection of high-risk RCC patients who

may benefit from treatment with novel molecular targeted

therapies, and serve as an appropriate biomarker by which

to measure tumor response to such agents. Indeed, sorafe-

nib and sunitinib, both of which are multitargeted tyro-

sine kinase inhibitors (TKIs) of the PDGF-R and VEGF-

R, are indicated for use in RCC.8,54 Support for this

notion is gained from studies demonstrating that in cancer

patients treated with targeted TKIs, tumor tissue exhib-

ited lower levels of pERK post-treatment. Specifically, sig-

nificant correlations between reduced pERK and disease

stabilization or partial responses were observed after ther-

apy with sorafenib55 and erlotinib56 in patients with met-

astatic carcinomas of the head, neck,55 and colon,56

respectively.

In summary, the results of the current study indicate

that pERK-1/2 is an independent prognostic biomarker

that significantly predicts the onset of metastasis in clini-

cally confined RCC. In addition, we propose that pERK-

1/2 may be used to better select patients with renal cancer

who may benefit from treatment with specific molecular

targeted therapies and/or as a tumor marker to measure

and monitor treatment efficacy when using such agents.

Further validation is required in other patient cohorts.

Conflict of Interest Disclosures

The authors made no disclosures.

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Activated extracellular signal-regulated kinase is an independent prognostic factor in clinically confined renal cell carcinoma