Large-scale independent validation of the nuclear factor-kappa B p65 prognostic biomarker in prostate cancer

https://doi.org/10.1016/j.ejca.2013.02.026Get rights and content

Abstract

Purpose

Over the last decade, we and others have uncovered a robust association between the nuclear localisation of nuclear factor-kappa B (NF-κB) p65, prostate cancer (PCa) aggressiveness and biochemical recurrence (BCR). Our goal was to validate these results in a large independent cohort of PCa patients who underwent radical prostatectomy.

Experimental design

A set of 1826 fully annotated prostate cancers treated by radical prostatectomy were analysed in a tissue microarray (TMA) format for NF-κB p65 immunohistochemistry-based protein expression. We performed standard Cox proportional hazard regression models for follow-up data, bootstrap procedure for model internal validation, Harrell’s concordance index for model discrimination and graphical assessment of predicted versus actual outcomes for model calibration.

Results

We observed a significant association between an increase in the nuclear frequency of NF-κB p65 and Gleason score (P < 0.001), overall BCR (P < 0.001) and development of metastases (P = 0.001). NF-κB was found to be an independent predictor of BCR (P < 0.001, Cox regression). However its contribution to the predictive accuracy of a multivariate model, which included preoperative PSA, Gleason score, extraprostatic extension, lymph node invasion, seminal vesicle involvement and surgical margin status, was modest.

Conclusions

Our study offers validating results linking NF-κB p65 with disease progression using a large cohort of European men. However, the contribution of NF-κB to a post-surgical predictive model appears modest. Further validating work should focus on evaluating the contribution of NF-κB p65 in pre-treatment models.

Introduction

Prostate cancer (PCa) is the most frequently diagnosed cancer and the second leading cause of cancer-related deaths for North American and European men.1 For several years, clinicians have used nomograms to stratify the risk of biochemical recurrence (BCR). These prognostic tools, such as the Kattan nomograms,2 are composed of several clinico-pathological variables (pre-operative serum PSA levels, post-operative Gleason score, extraprostatic extension, seminal vesicle involvement, lymph node invasion, and positive surgical margins status). However, the predictive accuracy of these nomograms needs to be improved as patients with similar clinico-pathological parameters follow divergent clinical evolution. Biomarkers, individually associated with PCa biology, could thus complement the clinico-pathological parameters in the prediction of cancer progression and help to accurately classify patients at risk of BCR.

One promising PCa biomarker is the nuclear factor-kappa B (NF-κB) transcription factor. The NF-κB transcription factor family is composed of the p65, RelB, c-Rel, p50/p105 and p52/p100 subunits that function as homo- or hetero-dimers.3 Classical NF-κB dimers are kept inactive in the cytosol by inhibitors of NF-κB (IκB). Following activation, IκB kinases (IKK) phosphorylate IκB, which results in its degradation and allows for the nuclear translocation of NF-κB where it can both induce and repress the expression of several genes containing κB elements in their promoter regions. In a cancer setting, NF-κB is implicated in oncogenesis and in chemoresistance through its action as a transcriptional regulator for key players participating in the suppression of apoptosis, cell adhesion, proliferation, the innate and adaptive immune responses, cellular-stress response and tissue remodelling.4

In recent years, our group demonstrated that the activation of the NF-κB pathway, defined by the nuclear translocation of the NF-κB p65 subunit, was associated with bone metastasis and lymph node metastasis,5, 6 predictive of lymph node invasion,7 and correlated with an increase risk of BCR.8, 9 Independent studies from other groups have confirmed these results suggesting that the evaluation of the cytoplasmic expression10 and the nuclear localisation of the NF-κB subunits can be used as an indicator of its activity and as a biomarker of PCa progression.11, 12, 13 Our goal was to validate these results in a large independent cohort of PCa patients who underwent radical prostatectomy.

Section snippets

Patients

Patients selected for the creation of the tissue micro-arrays (TMAs) used in this study have been described previously in detail.14 Briefly, primary tumours following radical prostatectomy were obtained from 3261 patients. All patients were recruited following informed consent. BCR was defined as a postoperative PSA of 0.2 ng/ml and rising and the interval was calculated based on the date of the first value of 0.2 ng/ml or higher. Patients without BCR were censored at the last follow-up date. A

NF-κB p65 expression

Positive nuclear NF-κB p65 staining (referred as ‘NF-κB’ staining from now on) was observed in 862 of the 1826 analysed malignant cores (47.3%), whereas cytoplasmic NF-κB staining was detected in 1793 of the 1826 analysed cores (98.2%) (Fig. 2g). Since NF-κB nuclear intensity had approximately the same distribution as nuclear frequency, we chose to only report data using the nuclear frequency. We found that patients with nuclear NF-κB had significantly more aggressive disease than patients

Discussion

One of the challenges in the clinical management of PCa is the accurate prediction of tumour aggressiveness and disease progression. Molecular biomarkers offer the possibility to further stratify patients with similar clinico-pathological parameters. Our group, and others, have exposed the potential relevance of including NF-κB in the decision making process. Previous studies on NF-κB expression in PCa included between 30 and 287 patient samples and demonstrated an increased expression in

Conclusions

This study offers validating results demonstrating that NF-κB can predict the development of BCR in univariable and multivariable models. However, the inclusion of NF-κB had only a modest impact on the predictive accuracy of post-operative models. Additional validation studies are warranted to address the independent prognostic utility of NF-κB in pre-treatment models.

Grant support

This study was supported by the Québec’s Ministère du Développement économique, de l’Innovation et de l’Exportation (MDEIE), the Terry Fox Research Institute and a Sanofi fellowship award. POG, LL, L-MS, VF, A-MM-M and FS are researchers at the CRHUM, which receives funding from the Fonds de recherche Québec-Santé (FRQS). FS is supported by the Université de Montréal’s Chair in Prostate Cancer. POG received a studentship from the FRSQ and LL from the Canadian Institutes of Health Research

Conflict of interest statement

None declared.

Acknowledgement

The authors would like to thank the Université de Montréal Urology Associates.

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