Biomarkers in bladder cancer: Translational and clinical implications

Please citeOncol/Hem

ARTICLE IN PRESSONCH-1784; No. of Pages 39

Critical Reviews in Oncology/Hematology xxx (2013) xxxxxx

Biomarkers in bladder cancer: Translational and clinical implicationsLiang Cheng a,b,, Darrell D. Davison a, Julia Adams a, Antonio Lopez-Beltran c, Lisha Wang a,d,

Rodolfo Montironi e, Shaobo Zhang aa Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, IN, USA

b Department of Urology, Indiana University School of Medicine, Indianapolis, IN, USAc Department of Pathology, Cordoba University, Cordoba, Spain

d Department of Pathology, Fudan University Shanghai Cancer Center, Shanghai, Chinae Institute of Pathological Anatomy and Histopathology, School of Medicine, Polytechnic University of the Marche Region (Ancona), United Hospitals,

Contents

1. Introdu2. Prolifer3. Apopto4. Tumor

4.1. T

4.2. R4.3. T4.4. P4.5. F4.6. p4.7. N4.8. F

5. Growth5.1. F5.2. E5.3. V5.4. A5.5. B5.6. T5.7. E5.8. E

CorresponIN 46202, US

E-mail ad

1040-8428/$ http://dx.doi.o this article in press as: Cheng L, et al. Biomarkers in bladder cancer: Translational and clinical implications. Crit Revatol (2013), http://dx.doi.org/10.1016/j.critrevonc.2013.08.008

Ancona, ItalyAccepted 13 August 2013

ction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00ation markers (PCNA, Ki67, and MIB1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00sis markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00suppressor genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00P53 and cell cycle regulators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

4.1.1. TP53. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.1.2. p21 (WAF1/CIP1/CDKN1A) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.1.3. p16 (INK4/CDKN2A/MTS1). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.1.4. p15 (INK4B/CDKN2B) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.1.5. p27 (KIP1/CDKN1B) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

etinoblastoma gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00SC1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00TEN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00HIT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0063 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00F1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00EZ1/LZTS1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

factors and receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00GFR3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00GFR/ERBB1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00EGF. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00FGF/FGF1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00FGF/FGF2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00GFB1. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00PHA2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00RBB2 (HER2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

ding author at: Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, 350 West 11th Street, Indianapolis,A. Tel.: +1 317 491 6442; fax: +1 317 491 6419.dress: liang [email protected] (L. Cheng).

see front matter 2013 Published by Elsevier Ireland Ltd.rg/10.1016/j.critrevonc.2013.08.008

Please cite this article in press as: Cheng L, et al. Biomarkers in bladder cancer: Translational and clinical implications. Crit RevOncol/Hematol (2013), http://dx.doi.org/10.1016/j.critrevonc.2013.08.008

ARTICLE IN PRESSONCH-1784; No. of Pages 392 L. Cheng et al. / Critical Reviews in Oncology/Hematology xxx (2013) xxxxxx

6. Oncogenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 006.1. HRAS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 006.2. MDM2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 006.3. . . . . . . 6.4. . . . . . . 6.5. . . . . . . 6.6. . . . . . .

7. Hormo . . . . . . 7.1. Androgen receptor (AR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 007.2. Estrogen receptor (ER) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 007.3. Progesterone receptor (PR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

8. Cell adhesion molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 008.1. E-cadherin and integrins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 008.2. CD44 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 008.3. CD24 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 008.4. Tetranectin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

9. Vessel density . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 009.1. Microvessel density . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 009.2. Lymph vessel density . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

10. Telomerase (TERT). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0011. Miscellaneous markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

11.1. Hyaluronic acid (HA) and hyaluronoglucosaminidase 1 (HYAL1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0011.2. Microtubule-associated proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0011.3. E2F transcription factor 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0011.4. Mammary serine protease inhibitor (maspin) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0011.5. Multidrug resistance proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0011.6. Cyclooxygenase 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0011.7. Gelsolin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0011.8. Uroplakin III . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0011.9. CAIX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0011.10. HIF1A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0011.11. Others . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

12. Combined biomarkers and nomograms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0013. Limitations of immunohistochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0014. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

Conflict of interest/funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00Reviewers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00Biography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

Abstract

Bladder cancer is associated with high recurrence and mortality rates. These tumors show vast heterogeneity reflected by diverse morphologicmanifestations and various molecular alterations associated with these disease phenotypes. Biomarkers that prospectively evaluate diseaseaggressiveness, progression risk, probability of recurrence and overall prognosis would improve patient care. Integration of molecular markerswith conventional pathologic staging of bladder cancers may refine clinical decision making for the selection of adjuvant and salvagetherapy. In the past decade, numerous bladder cancer biomarkers have been identified, including various tumor suppressor genes, oncogenes,growth factors, growth factor receptors, hormone receptors, proliferation and apoptosis markers, cell adhesion molecules, stromal factors,and oncoproteins. Recognition of two distinct pathways for urothelial carcinogenesis represents a major advance in the understanding andmanagement of this disease. Nomograms for combining results from multiple biomarkers have been proposed to increase the accuracy ofclinical predictions. The scope of this review is to summarize the major biomarker findings that may have translational and clinical implications. 2013 Published by Elsevier Ireland Ltd.

Keywords: Urinary bladder; Urothelial carcinoma; Biomarkers; Prognosis; Tumorigenesis

1. Introduction

Urothelial carcinoma is the 5th most common cancer inindustrialized countries, accounting for approximately 5%

of all cancers [13]. The associated risk factors for bladdercancer include tobacco smoking, aromatic amine exposure,arsenic exposure, chronic infection with Schistosoma species,radiation therapy, and exposure to alkylating agents [4,5].MYC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cyclin D1 and D3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .PAX5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IMP3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ne receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

Please cite canceOncol/Hem

ARTICLE IN PRESSONCH-1784; No. of Pages 39L. Cheng et al. / Critical Reviews in Oncology/Hematology xxx (2013) xxxxxx 3

Low grade papillary tumors comprise approximately80% of bladder tumors. These tumors most commonlypresent as superficial exophytic papillary lesions [1]. Mostpatients (7tumors, 20%5% presenthat these urothelial hsubsequentto a papillaa high rateinvasive isrepeated re5-year survbeen reporpT1 tumorbiomarkersprogress an

Biomarking to assibiomarkersbiology of clinical tria(Fig. 1). Inalterations identified [of clinical fication, prresponse tolearned aboefficiently this reviewthat may btumor biolo

2. Prolifer

Prolifernuclear proPCNA actsin eukaryohomotrimestrand syntcell cycle. dependent superficial tumors thatcells labelepatients withe PCNA grading, tusion is maxcycle. The P5 to 92%, aradiation th

cancer with PCNA labeling index below 30% of cells did notrecur, whereas aneuploid cancer with PCNA index greaterthan 30% usually recurred [25]. PCNA expression correlated

nuclell as wody agg cell

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statuer canargulinoma ympha8 (45i67 laiated

[32].ssion

d for rhe onfic sur this article in press as: Cheng L, et al. Biomarkers in bladder

5%) present with superficial (pTis, pTa, or pT1) present with muscle invasive (pT2) tumors, and

t with metastatic tumors. It has been suggestedtumors arise from normal urothelium through ayperplastic change as a benign papilloma, with

angiogenetic responses and further progressionry neoplasm. Although most bladder cancers have

of recurrence, their inherent capacity to become quite low. Most of these tumors are treated bysections and intravesical chemotherapy, and theival rate is approximately 90%. Progression hasted to occur in 1020% of cases. Up to 50% ofs may progress [6]. There is an urgent need for

that can distinguish tumors with the potential tod metastasize [714].ers may stratify bladder cancer behavior, help-

gn patients to appropriate treatments. Molecular in bladder cancer also offer information on thethe disease, prediction of oncologic end points forls, and prediction of response to targeted therapies

recent years, many new markers associated withat the molecular level in bladder cancer have been7,1518]. Some of these have even shown promiserelevance in the areas of diagnosis, tumor classi-ognosis, and prediction of an individual patients

treatment (Fig. 2). However, much remains to beut how these and other biomarkers can be used

to improve the management of bladder cancer. In, we focus on immunohistochemical biomarkerse related to urothelial carcinogenesis and bladdergical behavior.

ation markers (PCNA, Ki67, and MIB1)

ating cell nuclear antigen (PCNA) is a nonhistonetein that acts as an accessory of DNA polymerase.

as a processivity factor for DNA polymerase tic cells. The encoded protein is functional as ar that helps increase the processivity of leadinghesis during DNA replication in S phase of theThis protein localizes in nuclei in a cell cycle-

manner. Study of PCNA expression in 104 primaryurothelial carcinomas of the bladder found that

recurred had a significantly higher proportion ofd for PCNA [19]. In the study by Cheng et al. of 73th urothelial carcinoma of the upper urinary tract,index was positively associated with histologicalmor stage and patient prognosis [20]. Its expres-imal during S phase and is closely linked to the cellCNA labeling index in bladder cancer varies fromnd is predictive of cancer recurrence, response toerapy, and survival [2024]. Diploid urothelial

with as we

antiberatindepelabeltissueof apPCN

Knucleof thof imlatedinvesin ad[323similing inbut nanaly

Mtivitycomm

labelobserof blin sitH&EMIB626a Ki6is a uflat le

Inexpremens

in alwas a

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Mcarcieral lin 31for Kassoc

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exprelowewas tspeciatol (2013), http://dx.doi.org/10.1016/j.critrevonc.2013.08.008

r: Translational and clinical implications. Crit Rev

ar morphometric findings in bladder cancer cellsith MIB1 results [26,27]. MIB1 is a monoclonalainst the Ki67 nuclear antigen expressed in prolif-s. The MIB1 antibody binding appears to be lesson fixation parameters than PCNA, and reliablyferating cells in formalin-fixed paraffin-embeddedo studied in flash frozen bladder tumors, the ratios (seen in 90% of Ta and T1 bladder cancers) to

greater in patients without recurrence [28].a monoclonal antibody that recognizes a humanigen expressed in the S, G1, G2, and M phasescycle. Ki67 expression, measured as the numberreactive cells in frozen tissue specimens, corre-cancer grade and stage [2931]. A number of

rs have reported a prognostic role for Ki67 indexd urothelial carcinoma of the urinary bladderymph node metastases had expression that washat in the primary cancer [35]. Also, Ki67 label-as predictive of urothelial carcinoma recurrence,ays as an independent variable by multivariate

1,3640]. monoclonal antibody that displays immunoreac-rmalin-fixed paraffin-embedded sections, is most

used in clinical applications to determine the Ki67ex because of its convenience and favorable inter-nsistency [41]. Gunia et al. investigated 32 casescarcinoma in situ and 31 cases of noncarcinomalesions of the bladder by the mitotic counts on

ed sections and immunohistochemical staining forcinoma in situ had a Ki67 labeled percentage ofereas the noncarcinoma in situ flat lesions showedling of 3037%. The authors concluded that Ki67djunct in the differential diagnosis of challenging

of the urinary bladder [42].y of 226 patients with urothelial carcinoma, Ki67

was elevated in 43% of the cystectomy speci-4% of metastatic lymph nodes, but it was absentn cystectomy specimens [34]. Ki67 expressionted with advanced pathologic stage, higher grade,

cular invasion, and metastases to lymph nodes.s may also predict both disease recurrence andcer-specific mortality.s et al. assessed Ki67 expression in 713 urothelialpatients treated with radical cystectomy and bilat-denectomy at six centers. Bladder cancer recurred%) of these patients. Using a cutoff of 20%beled cells, the Ki67 positivity was significantlywith an increased probability of disease recur-

Ki67 labeling index was also associated with p53 [43]. In a multivariate analysis of 62 patients fol-ecurrent grade 1 cancer, MIB1 immunopositivityly significant predictor of recurrence and cancer-vival when compared with p53, HER2, and BCL2

Please cite this article in press as: Cheng L, et al. Biomarkers in bladder cancer: Translational and clinical implications. Crit RevOncol/Hematol (2013), http://dx.doi.org/10.1016/j.critrevonc.2013.08.008


Fig. 1. Category and function of biomarkers. Tumors are frequently caused by multiple level molecular alterations, including nuclear acid, protein or metabolite.These alterations represent the nature of the disease at different stages, including the disease development, progression, response to the therapy, and clinicaloutcomes.

Fig. 2. Biomarkers represent the mechanisms related to the tumor genesis, development, behavior and response to the therapy. Genetic biomarkers demonstratethe molecular alterations in nucleic acid level including structural or regulatory defects. Pathway biomarkers exhibit the alteration of pathway level including theactivation of oncogene, inactivation of tumor suppressor genes, altered signal transduction, or changes of microsystem. Effect molecule biomarkers detect themolecules direct or indirectly associated to tumor behaviors including self sufficient growth signal, insensitive to anti-growth signal, neoplastic differentiation,tissue invasion and metastasis, evading apoptosis, and sustained angiogenesis (Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000:100;57). All ofthe biomarkers need to be clinical relevant and have the potential to provide a better understanding of disease molecular mechanisms and provide aids to patientmanagement.



expression [44]. The multivariate analysis, however, did notinclude histomorphologic or clinical variables.

Low Ki67 labeling index was also associated withprogressionurothelial bcantly correwith a labethan those increased w

To prostional progp53 and Kirisk nonmuseries of p(BCG) [47predictor odent predicanalyses.

AssessinKi67 permtumor sampsame site. tion of resprecurrence

evaluation However, elimits the vof these coing Ki67 latechnique, Differencesprotocol, inanalysis cacohorts forand evidenand scorinuseful biom

3. Apopto

BCL2 imembrane cell prolifebenign andlial carcino[31,43,48and with hiunable to also did nopatients, reradiotheraprecurrence

Wild tymutant TP5discussion

is expressed more frequently in low grade, low stage urothe-lial cancer, whereas mutant p53 is more frequently found inhigh grade, high stage cancer. One possible explanation is

utanttic defally amay bcells,

nother (BCLendennot c

Most3%)

-XS (-XS celial ctic signBCG rs [56

(pT1 ficantlsis sh2 are tS (C

ligandhe so

e and sines. Uresencin patiimmunign

eased r stageaspaseple prays. I

noma.develurothspase

nced ph nodse, loctor omy [5

patiese-3 pancer

elial closs oadvanstasis redict this article in press as: Cheng L, et al. Biomarkers in bladder

-free survival. In a study of 309 patients with pT1ladder cancer, Ki67 was the only marker signifi-lated with progression-free survival [45]. Patients

ling index greater than 30% had a worse prognosiswith a lower index [27]. The labeling index alsoith increasing depth of muscle invasion [46].

pectively evaluate the prognostic utility of tradi-nostic factors and the proposed molecular markers67, Oderda et al. studied 192 intermediate and highscle invasive bladder cancers in a homogeneousatients treated with Bacillus Calmette-Gurin]. The labeling index of p53 was a significantf tumor recurrence and Ki67 was an indepen-tor of survival in both univariate and multivariate

g the proportion of cells staining for nuclearits pathologists to compare proliferation betweenles or between biopsies at different times for the

Such estimates are useful for prognosis, predic-onsiveness or resistance to therapy, estimation ofand progression risk after standard therapy andin real time of neoadjuvant therapy effectiveness.normous variation in analytical practice markedlyalue of quantitative immunohistochemistry in eachntexts. The many sources of variance in measur-beling index, even with the aid of digital imagingtherefore, remain a limitation of this biomarker.

in preanalytical specimen handling, in staining analytical methods, and in postanalytical datause discordance between institutions or patient

clinical studies. There is a need for harmonizedce based procedures in assessment, interpretationg of Ki67 proliferation fractions to make this aarker in clinical practice.

sis markers

s a protooncogene that encodes a mitochondrialprotein blocking apoptosis without influencingration. BCL2 immunoreactivity was observed in

dysplastic urothelium and in up to 80% of urothe-ma cases, but was negative in carcinoma in situ51]. BCL2 expression decreased with higher stagegher grade, although other investigators have beenreplicate this finding [31,43,48,52]. Expressiont correlate with prognosis for surgically treated

gardless of age [53]. For those treated with curativey, BCL2 immunoreactivity correlated with higherrate and with lower disease-specific survival [54].pe TP53 leads to apoptotic cell death, whereas3 inhibits apoptotic death, similar to BCL2 (see

below). Thus, it is interesting to note that BCL2

that mgeneclinicsion type [31].

ABAXindepdoes [55].(527BCLBCLurothgnosafter cance

sion signianalyBCL

FAFAS sis. Tlysatcell lthe psion FAS in beDecrtumo

Cmultipathwcarciwith with of caadvalympdiseapreditectoin 34caspalial curothhad with metative patol (2013), http://dx.doi.org/10.1016/j.critrevonc.2013.08.008


TP53 prolongs survival of cells with establishedects, allowing them to become more unstable andggressive. Conversely, BCL2 increased expres-e an early event that prolongs survival of wildallowing them to acquire initial genetic defects

apoptosis marker is the proapoptosis protein,2-associated X protein), that appears to be ant predictor of survival [48]. This marker, however,

orrelate with apoptotic rates of bladder cancers cancers were immunoreactive for both BAXand either BCL-XL (81%) or, less frequently,29%) [48,54]. Expression of either BCL-XL ororrelated with high grade and advanced stagearcinoma [48]. Ajili et al. investigated the pro-ificance of BAX and BCL2 in terms of recurrence

immunotherapy in 28 nonmuscle invasive bladder]. Univariate analysis showed that superficial inva-stage), high nuclear grade, and BAX expressiony increased the risk of recurrence. Multivariateowed that stage, age, and expression of BAX andhe best independent predictors of recurrence.D95) is a death receptor that when activated by

initiates downstream signals to induce apopto-luble form of FAS has been identified in the cellupernatant of high grade urothelial cell carcinomarine-soluble FAS is an independent predictor for

e of urothelial carcinoma and for disease progres-ents with a history of nonmuscle-invasive disease.nohistochemical staining is decreased from 90%specimens to only 58% in cancer specimens.FAS expression is also associated with higher, grade, and disease-specific mortality [57].-3 is a protease that induces apoptosis by cleavingotein substrates triggering apoptosis by varioust has been implicated in progression of urothelial

Caspase-3 overexpression has been associatedopment of muscle-invasive disease in patientselial carcinoma in situ. On the other hand, loss-3 expression has been associated with moreathologic stage, higher grade, and more frequente metastasis. For patients with muscle-invasivess of caspase-3 expression was an independentf bladder cancer-specific survival after radical cys-7]. Burton et al. evaluated caspase-3 expressionnts with carcinoma in situ, of whom 41% of theositive cases developed muscle-invasive urothe-

[58]. Data from the study of Karam et al. on 226ancer patients showed that 49% of the tumors

f caspase-3 expression, and this was associatedced pathologic stage, high grade, and lymph node[59]. Loss of caspase-3 was an independent nega-or of bladder cancer-specific survival.

Please cit canceOncol/Hem


Tissue microarray study of 179 bladder carcinomas fromcystectomy specimens for active caspase-3, single-strandedDNA, p53, SCL2, SAX, and COX2, in correlation with clin-icopathologstranded Dobserved aresults sugmarkers murothelial ccle invadinof BCL2, (32%), 111of over 220sis, altered were all asrence, and

Survivincancer [61caspase assdisease-spemal urothethe standarand metastresponse toindependencontaining treatment [

In a studistry, survivsurvival, divival. For ssurvival timsurvival raimmunohisuated in 51cells with pthe survivinin high graThe survivdicting tumamong patmost casesradical cystsurvivin exdictor of din multivarstandard cl

Immunocarcinomasof 61% (49positive stastage, and was foundclinicopath

To explsis factor-r

death receptors (DR4 and DR5) to bladder cancer recurrence,Li et al. immunohistochemically investigated the expressionof TRAIL, DR4, and DR5 in 229 bladder cancer patients

Cytopted inctivelcreaseperatissionate anan indval.

mor

tumocts a er. Whloss o

er, usu

TP53

ere isadderthwayllular tly peostati

rise to

. TP53 is a y a tuof chrer of r suppion an3 alsoutatioth of cr contes, wh) of sxpresed a sxprese half

in, wh much53 mo

of mun of otspotlly mie this article in press as: Cheng L, et al. Biomarkers in bladder

ical factors, found that active caspase-3, singleNA, p53, SAX, and COX2 were more frequentlymong high grade and higher stage tumors. Thegested that alterations in interrelated apoptosisay play an important role in the progression ofarcinoma from superficially infiltrating to mus-g tumor [60]. Karam et al. found that expressioncaspase-3, p53, and survivin were altered in 73

(49%), 120 (53%), and 141 (64%), respectively, bladder cancer patients [59]. In univariate analy-expression of BCL2, caspase-3, p53, and survivinsociated with high probability of disease recur-with disease-specific mortality.

is another promising biomarker in bladder66]. Survivin is an antiapoptotic inhibitor ofociated with aggressive disease, recurrence, andcific mortality. Survivin is not expressed in nor-lium [67]. Cisplatin-containing chemotherapy isd treatment for patients with locally advancedatic urothelial carcinoma of the bladder, but the

treatment rate is only 50%. Survivin is a strongt prognostic factor for poor response to cisplatin-chemotherapy and decreased survival after such68].y of 124 bladder cancers by immunohistochem-in expression significantly correlated with overallsease-specific survival, and progression-free sur-urvivin negative and positive tumors the medianes were 18.4 versus 9.8 months and the 5-year

tes were 28% and 5%, respectively [68]. Thetochemical expression status of survivin was eval-

bladder cancer patients. The percentage of cancerositive survivin nuclear staining has been termed

score. The survivin score was significantly higherde tumors (19%) than in low grade (5%) tumors.in score was superior to histologic grade for pre-or recurrence [63]. Disagreements of grading

hologists could be resolved by survivin scores in. Shariat et al. studied 726 patients treated withectomy and bilateral pelvic lymphadenectomy forpression [65]. Survivin was an independent pre-isease recurrence and of cancer-specific survivaliable analyses that controlled for the effects ofinicopathologic features.histochemical survivin staining of 80 urothelial

revealed nuclear and cytoplasmic positive scores/80) and 23% (18/80), respectively. Only nuclearining correlated strongly with increased grade,

probability of tumor recurrence. No relationship between the cytoplasmic survivin level and theological parameters [66].ore the interrelationship of human tumor necro-elated apoptosis-inducing ligand (TRAIL) and its

[69].detecrespeor inpostoexpretivariwas

survi

4. Tu

Aprotecanc

with canc

4.1.

Thof bllar paof cedirechomegive

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Th30 mhas ain TP95%regiothe husuaatol (2013), http://dx.doi.org/10.1016/j.critrevonc.2013.08.008


lasmic TRAIL, DR4, and DR5 expressions were 35%, 75%, and 74% of bladder cancer patients,y. Bladder cancer patients with either high DR4d DR5 expression had a significantly longerve recurrence-free interval than those with low

of both proteins during the 10-year followup. Mul-alysis revealed that expression of DR4 or DR5ependent prognostic indicator of bladder cancer

suppressor genes

r suppressor gene, or antioncogene, is a gene thatcell from one step in the multifactorial path toen a tumor suppressor gene undergoes mutationr reduction of function, the cell can progress toally by accumulation of other genetic changes.

and cell cycle regulators

strong evidence that malignant transformation urothelium results from alterations in molecu-s that are otherwise responsible for maintenancehomeostasis [70]. Alterations in these pathwaysrturb the regulation of cell proliferation and otherc mechanisms in urothelial cells that ultimately

the malignant phenotype.

353 kd DNA-binding phosphoprotein that is codedmor suppressor gene, TP53, located on the shortomosome 17 (17p13.1) (Table 1). It is the gate-the G1/S phase of the cell cycle and acts as aressor [71]. This transcription factor regulates celld inhibits cells from entering the S phase.

regulates antiapoptotic genes such as BAX. Lossn of TP53 results in unregulated and aberrantells whose proliferation would normally be kept

rol. Malats reviewed 168 publications from 117ich included 10,026 patients, and found that 27%tudies that assessed the prognostic value of p53sion in recurrence by use of multivariate testsignificant independent association between p53

sion and recurrence [72].-life of wild type p53 is estimated between 20 andereas, due to decreased degradation, mutant p53

longer half-life, estimated to be 24 h. Mutationsst frequently involve exons 511. Approximatelytations occur between exons 5 through 8 in the

the DNA binding domain, otherwise known as region of the TP53 gene. Mutations in TP53 aressense substitutions that cluster in one particular



Table 1Select mutations in urothelial neoplasms.

Gene(s) Chromosome location Frequency in bladder neoplasms Mechanism(s)P16INK4

FGFR3

ERBB2 (HERTP53

MDM2 EGFR HRAS RB1 mas FHIT SFRP1 PTEN DBC1 PTCH (GorlinTSC1 APC PIK3CA AKT1 CTNNB1 KRAS BRAF

a FGFR3 mb TP53 mut

region of t290, involv270286. Tand are proregion encTP53 mutatumors) andof all nonurmorphism peripheral risk of bladdictive of b

Loss of many humatributes to are deletederozygous oligomericcells transfcomplex re

Mutatedtransform endogenoution of chrochromosomis a late ereflecting ction of cancould lead

ivationin [78

approvancedr supp9p21 >50%

4p16.3a 74% pTa 21% pT116% >pT2>40% overall

2) 17q23 3750%, especially >pT2 17p13b 5060% high grade or invasive

carcinomas12q14 46%7p12.3p12.1 3050% invasive carcinomas 13q14.1q14.2 3040% 13q14.114.2 >50% high grade urothelial carcino3p 2560%8p 2530% 10q23 2030% 9q32-33 >50%

syndrome) 9q22 >50% 9q34 1234% 5q21 56% 3q26.3 2025% 14q32.3 5% 3p21 2% 12p12.1 412% 7q34 07%

utation is mainly seen in exon 7, 10, and 15.ation is mainly seen in exon 58.

he gene product between amino acids 130 anding residues 117142, 171181, 239258, andhese regions are highly conserved among species,bably necessary for normal TP53 function. The

ompassing codons 280 and 285 is a hot spot for

inactprote

Inin adtumo this article in press as: Cheng L, et al. Biomarkers in bladder canceatol (2013), http://dx.doi.org/10.1016/j.critrevonc.2013.08.008

tions in urothelial carcinoma (12% of high grade is not usually found in other malignancies (13%othelial malignancies) [73]. TP53 codon 72 poly-and homozygosity for arginine at residue 72 inblood leukocytes was associated with increasedder cancer, although this mutation was not pre-ladder tumor invasiveness [74,75].heterozygosity (LOH) of the TP53 locus occurs inn cancers [76]. Therefore, TP53 most likely con-human carcinogenesis when both normal alleles

or inactivated. Transforming activity in the het-state may also occur due to formation of an

complex between mutant and wild type TP53. Inormed with mutant TP53 gene, the altered proteinmains in the cytoplasm.

TP53 genes can cooperate with RAS genes toprimary cultured fibroblasts in the presence ofs wild type p53 protein [77]. In some cancers, dele-mosome 17 loci occurs simultaneously with otheral abnormalities, suggesting that TP53 mutation

vent in carcinogenesis. Cancer cell aneuploidy,hromosomal instability, may play a role in selec-cer cells with TP53 gene mutations. This process

to loss of the remaining wild type allele and

more comm

flat carcinotions have those withof bladder with mutantime in thoversus 51 ingly, tumopoorer prog

p53 proas the E6 pis detectedinvasive caHPV correlmutations acancer, sug

MutatioTP53 genesof normal half-life anmakes it imnuclei. Ovpoor prognInactivation of CDKN2 leading to uncontrolled cell cyclesignaling pathwaysActivation of RAS-MAPK pathway

Encodes for receptor protein tyrosine kinaseTumor suppressor gene

Regulation of protein degradationTyrosine kinaseOncogeneTumor suppressor geneTumor suppressor geneTumor suppressor geneTumor suppressor geneTumor suppressor geneTumor suppressor geneTumor suppressor geneTumor suppressorSignal moleculeSerine-threonine protein kinaseWnt pathway componentOncogeneOncogene

of growth control function of the normal p53].ximately 50% of bladder tumors, especially those

stages of disease, missense mutations in the TP53ressor gene are found [79]. Mutations of TP53 arer: Translational and clinical implications. Crit Rev

on (>50%) in high grade invasive tumors and inma in situ. Therefore, tumors with TP53 muta-

worse prognosis and a higher recurrence rate thanout the mutation. A comparison of the survivalcancer patients with wild type p53 versus thoset p53 showed a significantly decreased survivalse with mutant p53 (median survival, 12 monthsmonths for wild type p53) [80,81]. Not surpris-rs with p21 mutations are also associated with anosis and higher recurrence rate.

tein can also be inactivated by viral proteins suchrotein of human papillomavirus (HPV) 16. HPV

in occasional cases of papillary noninvasive andncer (12% in one study), and the presence ofates with higher stage and grade [74,8286]. TP53re rarely observed in patients with HPV-positivegesting separate etiologic pathways [82].ns of TP53 or functional inactivation with intact

are common in many human cancers, causing lossgrowth regulation. Mutations result in prolongedd accumulation of the p53 protein to a level thatmunohistochemically detectable in cancer cell

erexpression of p53 protein is associated with aosis in a variety of cancers and appears to precede



loss of chromosome 9 in carcinoma in situ as a precursor ofinvasive bladder cancer [87].

Most antibodies for p53 require antigen retrieval proce-dures whenStaining reimen pretreof differentrely upon thalf-life inimmunoreamutations the setting damage. Inin protein aimmunohisent of p53 extent of thcorrelationsubstitutionplays stainis observedthresholds of cells, 10Intratumortheless, theexpression[35].

The cellanalysis ofmutations mutations metastasesmary cance

In a stumas, altereand was indcancer-spep53 can hepatients intwith data fCancer (ISing resultsanalysis of an indepenysis regard

TP53 alincreased sage DNA, In patientsresulted in fold increaabout 9 yeano survivalgest that p(those withbenefit from

identify patients who should receive postoperative cytotoxicchemotherapy and eliminate those for whom such treatmentwould be futile. Nonetheless, the response of tumors with

mutac agenuding ture [7linicalurethr

urothity cor

and p7p delunoreany rep

in ot T1

unoreaith few

respe than ion ra

per ytudy sendenCL2 er ca

n D1, ycle pclin Ding indrs.e pre

withploidyn, preder [117een p5de clibout 6tions, ypia amutati, and

arcinomissio

includnts car

mutaers, an

substit of 1

of Tbe praddertions e this article in press as: Cheng L, et al. Biomarkers in bladder

used with deparaffinized formalin-fixed sections.sults may vary due to differences in fixation, spec-atment, antibody binding site exposure, and use

antibody clones. Immunohistochemical methodshe accumulation of p53 protein due to prolonged

cells with TP53 missense mutations. p53 nuclearctivity is usually but not always indicative of TP53[35]. Wild type p53 protein may accumulate inof TP53 activation, including hypoxia and DNA

addition, not all TP53 missense mutations resultccumulation and these may cause false-negativetochemical results. Finally, there may be a gradi-inactivation that varies according to the site ande mutation. Nonetheless, there is a strong positive

between immunoreactivity and TP53 missense mutations [88,89]. Benign urothelium rarely dis-

ing, whereas p53 staining in urothelial carcinoma in 1878% of cases [9093]. Different detectionhave been used for positive staining, including 0%% of cells, and 20% of cells [31,43,90,91,9398].al heterogeneity should also be considered. Never-

investigators in one study found no difference in between the central cancer and the invasive front

ular urine sediment may also be used for genetic TP53 mutations [99,100]. Comparison of TP53in microdissected cancer correlates poorly withobserved in urine and blood [101]. Lymph node

have expression that is similar to that in the pri-r [35].

dy of 80 patients with pT1N0 urothelial carcino-d p53 expression was found in 25% of patientsependently associated with cancer recurrence and

cific mortality [102]. The authors concluded thatlp stratify the heterogeneous population of pT1o different risk groups. These findings were in linerom the International Study Initiative on BladderBC) combining immunohistochemical p53 stain-

from 26 different studies [103]. This combined929 patients with T1 bladder cancer demonstrateddent prognostic value of p53 in multivariate anal-ing progression, but not overall survival [103].terations in urothelial carcinoma may result inensitivity to chemotherapeutic agents that dam-including doxorubicin and cisplatin [104,105].

with TP53 mutations, adjuvant chemotherapya threefold decrease in risk of recurrence and a 2.6-sed chance of survival with median followup ofrs [104]. Patients without TP53 mutations derived

advantage with chemotherapy. These results sug-atients at greatest risk of progression and death

TP53 mutations) may also derive the maximum adjuvant chemotherapy. Thus TP53 status may

TP53peuti(inclliteratial ctrans

Inactivrence

ing 1Immin maputedStageimmcer w

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tions to various therapies, including chemothera-ts, radiation therapy, and DNA-damaging agents

cisplatin and doxorubicin) has been variable in the7,106108]. TP53 status was not predictive of ini-

response to BCG therapy in T1 cancer treated byal resection, regardless of grade [109,110].elial carcinoma, nuclear p53 protein immunore-related with high grade, high stage, cancer recur-rogression, survival, and TP53 mutations, includ-etion and 17 polysomy [31,43,53,91,97,111131].ctivity had independent prognostic significanceorts [91,95,120,125,132,133]. This has been dis-

her reports [31,92,94,112,113,117,122,134136].bladder carcinoma with more than 20% p53ctive cells had a higher progression rate than can-er stained cells (21% versus 3% progression per

ctively) [132]. Similarly, carcinoma in situ with20% p53 immunoreactive cells had a higher pro-te than cases with fewer stained cells (86% versusear, respectively) [91,133,137139]. Conversely,howed that cancer grade and stage were the onlyt predictive factors for patient survival when p53

were included in the analysis [31]. A study of 491ncers for aberrant expression of p53, p63, p16,RB, and Ki67 revealed significant aberration ofroteins p53, p63, p16, and Ki67, but not of RB1 [140]. Overexpression of p53 and high Ki67ex were associated with high stage or high grade

dictive value of p53 may be increased when com- other factors. p53 immunoreactivity and DNA

are closely associated, and, when found in combi-ict a very poor outcome for patients with invasive]. Conversely, another study found no correlation3 expression and DNA ploidy status, but did not

nical outcome data for the patients [118].5% of cases of carcinoma in situ contain TP53considerably greater than the 2833% of casesnd dysplasia [141143]. This high frequency ofon is similar to that of invasive urothelial carci-may explain on a genetic basis the great propensityma in situ to progress [141]. Moreover, germlinen of TP53 mutations occurs in cancer-prone fam-ing those with Li-Fraumeni syndrome, and thesery a high risk of urothelial carcinoma [144]. TP53tions were present in 11 of 18 invasive bladderd the most common mutation was single baseution [145]. Missense mutations were present in1 cases, and nonsense mutations in three. Muta-P53 are also detectable in urine sediment andedictive of progression [145,146]. In a study of

cancers from 23 patients, the incidence of TP53was significantly higher in muscle-invasive than



nonmuscle-invasive cancer (58% versus 8%, respectively)[129]. High grade bladder cancer contained diverse TP53mutations in 3651% of cases [77,106]. These molecularstudies conp53 proteicarcinoma.

The ideurothelial cthat similardifferent gebelow) [14

4.1.2. p21 p53 ind

class of gekd protein ble for initwith loss owhich is aTP53 geneloss of cycG1 [76]. Tkinase inhibasic enzyregulation.

p21 genp53-dependthere was expressioncisplatinumvival than 23 monthspredicted cinvasive cacombinatioin patients therapy; thwhereas thauthors posmutant p53pathway by

In an imsion in 242was an indvival after et al., includer, proteinwith cancealtered exprisk of bladsuggestingin these pap27 stratifisignificantlgression [1carcinoma,

recurrence and cancer-specific mortality [152]. In patientswith organ-confined disease, p21 remained independentlyassociated with disease recurrence and cancer-specific death

com

[153].ound n inder canc

. p16 smallcontro, by core regated adder c

16 genon, weadder

9, is noma ention

anom

estingancer [

NME withlated 162].verexprs, co

eased oor p165].

ficantl T1 p

ng as 16 exignificbility

ed 73 icallyof casessionaton eogy stypica

immused in

in 37ich 3, the ity in ding lsions

cytol this article in press as: Cheng L, et al. Biomarkers in bladder

firm the immunohistochemical observations ofn expression for predicting outcome in bladder

ntification of p21 and p16 gene mutations inarcinoma and other human cancers has shown

biologic effects can be caused by alterations ofnes in the p53 regulatory pathway (see discussion7,148].

(WAF1/CIP1/CDKN1A)uces TP53-dependent genes. A prototype of thisne, p21 (WAF1/CIP1/CDKN1A), encodes a 21that inhibits cyclin-dependent kinases responsi-iation of the G1 phase of the cell cycle. Tumorsf TP53 function show downregulation of p21,

downstream target of TP53. Mutations in the result in failure to stimulate p21, with subsequentlin-dependent kinase inhibition and initiation ofhe discovery of p53-dependent cyclin-dependentbitors linked this tumor suppressor gene to theme mechanisms operative in normal cell cycle

e is transcriptionally activated by p53 and mediatesent G1 arrest following DNA damage. Althoughno apparent association between p53 and p21, patients with p21-positive cancers receiving-based systemic chemotherapy had greater sur-p21-negative cancer patients (60 months versus, respectively) [122]. Similarly, p21 expressionancer-specific survival in patients with muscle-ncer treated by radiation therapy [54,128]. Then of p21 and p53 improved prediction of survivalwith muscle-invasive cancer treated by radiationose with p21+/p53+ cancer had the best survival,ose with p21/p53+ had the worst prognosis. Thetulated that p21/p53+ patients may accumulate

protein and yet have derangement of the p53 loss of the critical effector molecule, p21 [149].munohistochemical study of p21 and p53 expres-

patients with urothelial carcinomas, p21 statusependent predictor of tumor recurrence and sur-radical cystectomy [108]. In the study of Shariatding 49 patients with carcinoma in situ of blad-

expression of p21 was independently associatedr recurrence and progression [150]. Patients withression of both p53 and p21 were at greatestder cancer recurrence, progression, and mortality,

a potential rationale for early definitive therapytients. Combination of p21 with p53, pRB, anded patients with nonmuscle-invasive disease intoy different risk groups for recurrence and pro-51]. In patients with muscle-invasive urothelial

p21 was an independent predictor of both disease

whenp53 tors fnot aor fo

4.1.3A

p16, to Mthat ais locin blathe ptivatiof blsome

carciconv

over,

suggder cgenelationcorre

[161,O

cance

Decrand p[163signiTa orsentiof pwas s

probastudichem54% progr

Picytol19 acases

asses

foundof whstudyactivinclulial leurineatol (2013), http://dx.doi.org/10.1016/j.critrevonc.2013.08.008


bined into a multiparameter test with p27 and However, another study by the same investiga-in 80 patients with T1 disease only that p21 waspendent predictive marker for disease recurrenceer-specific mortality [102].

(INK4/CDKN2A/MTS1)er TP53-related cyclin-dependent kinase inhibitor,ls cell progression from G1 phase to S and from G2ntrolling cyclin-dependent protein kinases (CDK)ulated by cyclins D, E, and A [154]. The p16 generound 9p21, which is a major site for deletionsancer [155,156]. Deletions and/or methylation ofe promoter CpG island, with consequent p16 inac-re also reported as early events in tumorigenesis

cancer [157]. The p16 gene, present on chromo-abnormal in up to 60% of cases of squamous cellassociated with Schistosomiasis, but only 18% ofal urothelial carcinoma cases [158160]. More-alies of p16 and TP53 are mutually exclusive,

a complementary role in the pathogenesis of blad-159]. Synchronous p53 and metastasis suppressor1 (NM23-H1) detection showed significant corre-

poor patient survival, although NME1 by itselfwith extent of cancer invasion and recurrence

ression of p16 was observed in 4051% of bladdermpared with absence in benign urothelium [163].expression correlated with increasing grade, stage,rognosis, although the opposite was also reported

A recent study found that p16 expression wasy higher in muscle-invasive cancer that followedrimary cancer when compared with cancer pre-muscle invasive at first diagnosis [166]. Loss

pression was found in 33 patients (59%) andantly associated with the reduced recurrence-free

[167]. This was confirmed by Kruger et al., whosamples of T1 bladder carcinomas immunohisto-

for p16. Loss of p16 expression was observed ines and was significantly associated with reduced-free survival [168].t al. investigated p16 expression in 84 urinaryamples including 18 reactive, 10 low grade,l urothelial cell, and 37 high grade carcinomanohistochemically [169]. Positivity for p16 was

75/84 (89%) urinary cytology cases and was/37 (100%) of the cases with high grade cytology,5 (97%) were confirmed by histology. In anotherauthors compared the results of p16 immunore-cytology and in biopsy samples from 83 cases,ow grade urothelial carcinomas, reactive epithe-, and negative cases [170]. The p16 expression inogy samples showed a sensitivity of 67% and a

Please cit cancer: Translational and clinical implications. Crit RevOncol/Hem


specificity of 83% in the diagnosis of low grade urothelialcarcinomas. Nakazawa et al. compared the p16 expressionimmunochemically in 116 urine cytology samples [171].Overexprescinomas, infor detectio

4.1.4. p15This tum

encodes a c9p21. Exprlium, decrin muscle-region conbladder ursignificantlcial (TaT1deleted frostatus wereof the bladthe p16 ander tumorswith lower tosoma endto high tum

4.1.5. p27p27 is

dependent cyclin D acycle arresfor p27 in cinoma, hoincreased pwith recurmortality [

Loss ofincreased anoma in siof invasivep27 in the nopment of invasive blnuclear p27by immuno

4.2. Retino

The retencodes a adult cells product supprogressionthe RB geuct is inacHPV16 wi

Table 2Select molecular biomarkers in bladder cancer.

Proliferation markersliferating cell nuclear antigen (PCNA)71

osis markersL2Xpase 3 (CASP3)vivinersr suppressor genes, oncogenes, mutator genes, and cell cyclelators3

(WAF1/Cip1/CDKN1A) (INK4/CDKN2A/MTS1) (INK4B) (KIP1)inoblastoma gene (RB)1N

gile histine triad gene (FHIT)3

ASM2Clins D1 and D3 (CCND1 and CCND3)53

ersh factors and receptorsroblast growth factor receptor 3 (FGFR3)dermal growth factor receptor (EGFR)cular endothelial growth factor (VEGF)dic fibroblast growth factoric fibroblast growth factor (bFGF)nsforming growth factor (TGF) beta

B2/HER2ersone receptorsrogen receptorogen receptorgesterone receptordhesion markersadherin4424acectiners

densityrovessel densityph vessel density

erasellaneous protein markersltidrug resistance proteinslooxygenase 2 (COX2)solinocrine motility factorinal epithelial antigen (LEA.135)

kinase-type plasminogen activator factorface glycoprotein T138luronic acidoxalase system enzymes1/LTS1 tumor suppressor gene15/BTAK/Aurora A gene producte this article in press as: Cheng L, et al. Biomarkers in bladder

sion of p16 was detected in 80% of high grade car-dicating the usefulness of p16 as an ancillary tooln of urothelial carcinoma with high malignancy.

(INK4B/CDKN2B)or suppressor gene found adjacent to the p16 geneyclin-dependent kinase inhibitor on chromosomeession of p15 mRNA was present in benign urothe-eased in superficial cancer, and heterogeneousinvasive cancer [172]. The 9p21 chromosomaltaining this gene is frequently deleted in humanothelial carcinoma. p15 mRNA expression wasy decreased in 66% of 86 patients with superfi-) bladder cancers. In 50% of these, p15 gene was

m the tumor cell genome [172]. p16 and p15 DNA studied in 110 patients with urothelial carcinomader. Homozygous deletion (both alleles lost) ofd the p15 genes was observed in 11 and 9 blad-, respectively, and these deletions were associatedtumor stage [159]. In bladder tumors from a Schis-emic region, deletion of both genes was relatedor grade and presence of bilharziasis [173].

(KIP1/CDKN1B)a member of the CIP/KIP family of cyclin-kinase (CDK) inhibitors, which act to inhibitnd cyclin E kinase activity. This results in cellt in the G1 phase. There is limited predictive valuepatients with nonmuscle-invasive urothelial car-wever in patients with muscle invasive disease,27 immunohistochemical staining is associated

rence, disease progression, and disease-specific57].

p27 expression in tumor nuclei coupled withctivated caspase 3 expression in bladder carci-

tu cells may predict the subsequent development bladder cancer [58]. Loss of immunostaining forucleus was associated with the subsequent devel-

invasive disease. In contrast, the patients in whomadder cancer did not develop had high levels of

but low levels of activated caspase 3 expressionstaining.

blastoma gene

inoblastoma (RB) gene on chromosome 13q14105 kd protein that regulates transcription in all(Table 2 ). The normal hypophosphorylated genepresses expression of genes required for cell cycle. Cyclin and cyclin-dependent kinases inactivate

ne product by phosphorylation. RB gene prod-tivated by the protein coded in the E6 gene ofthout mutation of the RB gene [174]. The p16

ProKi6MIB

ApoptBCBACasSurOth

TumoreguTP5p21p16p15p27RetTSCPTEFraTP6HRMDMYCycPAXIMPOth

GrowtFibEpiVasAciBasTraERBOth

HormAndEstrPro

Cell aE-cCDCDTetrOth

VesselMicLym

TelomMisce

MuCycGelAutLumUroSurHyaGlyFEZSTKatol (2013), http://dx.doi.org/10.1016/j.critrevonc.2013.08.008



Table 2 ( Continued )

Peroxisome proliferator-activated receptor gammaTissue polyThymidylaThymidinefactor)DihydropyMatrix metTissue inhiProline-dirClusterinOsteonectinKu proteinCaveolin 1GlycolipidsHypoxia inS100 calciuBladder canCathepsinsMAGEA4 Oxygen-regHepatoma Others

gene encodtein, is an uits suppresscorrelates whaps accouabsence or

Any typinhibition oprogressioncell prolifegrade urothtions [176,invasive bl

RB is extion of RBretinoblastoof alteratiotion. Majorof a propeincluding nate impropcodons, amthat destabotherwise cause absenin bladder rather thangenetic facof high grof RB funcnonpapillarwith LOH cle invasiosimilar to t

Altered expression of pRB is associated with decreasedsurvival of patients with urothelial carcinoma [180]. Benign

elial mucosa and noninvasive urothelial carcinoma haveimmuical dncer

e p53,nohis

for R% of rs, buent w

TSC1

e TSeat srtin a

is lo4% o

is gen mutat1871ladder

a wor

r progssion

wholenctionation, SC1

itor, e in 1

r TSC[192]

or hanohispeptide-specific antigente synthase

phosphorylase (platelet-derived endothelial cell growth

rimidine dehydrogenasealloproteinase 1 (MMP1)bitor of metalloproteinase (TIMP)1ected protein kinase F(A)

(CAV1) and glycosyltransferases GM3 synthaseducible factor (HIF) 1 alpham binding protein A4 (S100A4)cer-associated protein (bc10)

proteinulated protein (ORP150)

upregulated protein (HURP)

ing the p16 cyclin-dependent kinase inhibitor pro-pstream regulator of RB maintaining the pRB inive or hypophosphorylated state. Absence of p16ith functional inactivation of the RB protein, per-

nting for the equal prognosis in patients with RB1overexpression in some studies [175].e of mutation of the RB gene may lead to decreasedf the E2F transcription factor family required for

through G1 and S phases, leading to increasedration. It has been found that over 50% of highelial carcinomas have both TP53 and RB muta-

177]. E2F3 is amplified in approximately 14% ofadder cancers [178].pressed in all human tissues. Mutational inactiva-

gene and reduction of pRB expression occurs in

urothpRB chemof caunlikimmutionsin 32cance

sedim

4.3.

Ththe hhamaTSC1that 3in thgene[185,

Bhaveand/oexpre

Aof fuactivand TinhibTSC2eithe15% ativeimmu this article in press as: Cheng L, et al. Biomarkers in bladder canceatol (2013), http://dx.doi.org/10.1016/j.critrevonc.2013.08.008

ma and other cancers [179]. The two main typesn of RB in human cancer are deletion and muta-

deletions of large gene segments result in absencerly functioning gene product [179]. Mutations,ucleotide substitutions alter gene function, cre-

erly located initiation signals, splicing sites, stopino acid substitutions, and induce other changes

ilize transcription, truncating the gene product ormodifying the messenger RNA. These changesce of functional pRB protein [180]. RB alterationscancer usually appear as a subtle point mutation

a major deletion. The RB gene is one of the majortors responsible for development and progressionade muscle-invasive bladder cancer [174]. Losstion occurs in 30% of high grade papillary andy urothelial carcinomas. Loss of RB correlates

at the RB gene locus, high tumor grade, and mus-n. Lymph node metastases have pRB expressionhat in the primary cancer [35].

ity of recurTSC1 LOHray study showed thato have a his an indepinterval of TSC1 has aulating thethe cyclin-d

4.4. PTEN

PTEN (chromosomof LOH in[188,1951in the samto decreasir: Translational and clinical implications. Crit Rev

noreactivity in most cells [181]. Immunohisto-etection of pRB appears to be a useful markerprogression, but is not routinely used because,

abnormal pRB results in decrease or variability oftochemical staining [174,182,183]. Allelic dele-B and MYCL1 in urine sediment were observedcases of carcinoma in situ and in 20% of bladdert the correlation between cancer tissue and urineas not strong [184].

C1 gene (tuberous sclerosis complex 1) encodeshock protein binding 1164-amino acid peptidend is mutated in patients with tuberous sclerosis.cated on 9q34 [185]. LOH studies have shownf bladder cancers, especially Ta, have mutationse [186]. Other investigators have reported TSC1ion frequency rates of 1113% in urothelial tumors89].

tumors with altered expressions of TSC1 tend tose prognosis and with more frequent recurrenceression in comparison with tumors having normal

[186,190]. genome sequencing experiment found that loss

mutation in TSC1, a regulator of mTOR pathwaywas present in 8% of 109 bladder cancers studied,mutation correlated with sensitivity to the mTORverolimus [191]. Mutation analysis of TSC1 and45 cases of urothelial carcinomas showed that1 or TSC2 was mutated at a combined frequency of. Primary tumors with TSC1 mutation were neg-d significantly reduced expression for TSC1 bytochemistry [193]. In another study, the probabil-rence was significantly increased in patients with

[186]. An immunohistochemical tissue microar-of 134 pTa and pT1 bladder tumors for TSC1t reduced or absent expression of TSC1 tendedigher risk of progression. Low TSC1 expressionendent factor for predicting shorter progressionpTa/pT1 tumors. In a subset of pTa/pT1 tumors,

role in bladder carcinogenesis by positively reg- p53 effector 14-3-3 protein sigma (stratifin) andependent kinase inhibitor p27 [190,194].

phosphatase and tensin homologue deleted one 10) is located on 10q23, a common region

high grade and high stage urothelial carcinoma97]. Loss of PTEN causes PI3 kinase activation

e way as TSC1. Overexpression of PTEN leadsng cell growth and tumorigenicity in vitro and



in vivo [198]. Fifty-three percent of primary bladder cancersexhibited decreased or absent expression of PTEN proteinin either the cytoplasm or nucleus of tumor cells [199]. Inadvanced breduced, padecrease ingrade [199

The clinassociationdecreased to tumor ssurvival in histochemicancer sam

PTEN expcal stage, gcarcinomaspromise fo

4.5. FHIT

Fragile arm of chrexpressionidentified imore comm

whose cancnoses and models sho/ and +/mice develbutyl-N-(4protein expgrowth.

Baffa etsix bladderin 67% of tImmunohislial cancersreduced in

The rolegenesis of Zhang et alurotheliumFHIT protbetween nadvanced tsize, tumoassociationmarkers [2

4.6. p63

p63 is a normal urop63 (/)

[210]. p63 has been implicated in maintenance of epithelialstem cell compartments [211]. Immunohistochemical studiesdemonstrated that p63 is downregulated in muscle-invasive

er canval in ion of

in micselectefore, tenanctem ce

tissuest inv

rficial of p6as fo

w malarcinoive tu

NF1

e neuator ofibromF1 ge

in is . The lrolife

no m

later rease noma pigen

exprealtonession. Redu

mor sph gra

FEZ1

ucine1/LZTosom

ers of signifir mor

]. LOHt in thiated

proteelial cTS1

ancer e this article in press as: Cheng L, et al. Biomarkers in bladder

ladder cancers, PTEN protein was significantlyrticularly in the nucleus, in 94% of cases, and this

PTEN correlated with higher disease stage and].ical significance, as with most bladder biomarkers, was controversial [200,201]. Although PTENexpression was significantly different accordingtage and grade, it failed to predict recurrence or190 prospectively studied patients [201]. Immuno-cal analysis of PTEN expression in 989 bladderples demonstrated that the pathologist scoring ofression negatively correlated with the pathologi-rowth pattern, and histological grade of urothelial, but other mTOR pathway markers also showed

r prediction of cancer specific survival [202,203].

histadine triad gene (FHIT) is located on the shortomosome 3 (3p14.2) and has decreased protein

in bladder tumors [204]. Loss of FHIT has beenn 2560% of bladder cancers, and this finding ison in high stage urothelial carcinomas. Patients

ers show this loss of expression have poorer prog-decreased survival [205207]. Animal knockoutwed that 8/28 (28%) and 6/13 (46%) of the FHIT mice, respectively, versus 2/25 (8%) FHIT +/+oped invasive carcinoma after treatment with N--hydroxybutyl) nitrosamine [208]. Restored FHITression induced apoptosis and inhibited tumor

al. reported deletions at the FHIT locus in 50% of carcinoma cell lines and loss of FHIT expressionhe six carcinoma cell lines by Western blot [206].tochemical assay of 85 primary bladder urothe-

revealed that FHIT protein was absent or greatly61% of the tumor tissues [206].

and clinical significance of FHIT in the patho-bladder urothelial carcinoma was investigated by., who found that 40/42 (95%) of normal bladder

and 59/25 (47%) of bladder cancers expressedein [209]. There was a significant associationegative FHIT expression in bladder cancer andumor stage, high pathological grade, large tumorr recurrence, and reduced survival time, but no

with age, gender, or number of positive tumor09].

member of the TP53 gene family that is present inthelium but lost in most invasive cancers [210].mice fail to complete urothelial differentiation

bladdsurviablatationwith Thermainlial susingin mosupestudycinomof lolial cinvas

4.7.

Thregulneuro

The Nprotecellscell pwere

ever,

a deccarcithat egene

Aexpremens

of tuin hig

4.8.

Le(FEZchromcanc

was

highe[208even

assoc

FEZ1uroth

LZder catol (2013), http://dx.doi.org/10.1016/j.critrevonc.2013.08.008


cers, and loss of p63 is associated with shorterpatients with bladder cancer [212,213]. Targetedp63 expression disrupts normal bladder differenti-e, leading to loss of the basal/suprabasal layer, but

ive retention of so-called umbrella cells [214].p63 is believed to play an essential role in thee of self-renewal and survival of normal urothe-lls [211]. Urist et al. examined 160 bladder tumors

microarrays and found that p63 expression is lostasive cancers (16%) but retained in most papillarytumors (93%) [210]. An immunohistochemical3, p53, and MIB1 expression in 158 urothelial car-und that p63 expression distinguished neoplasmsignant potential or noninvasive papillary urothe-ma low grade (93%) from high grade nonmusclemors (31%).

rofibromatosis-related protein (NF1) gene is af cell growth and differentiation whose product,

in, is an inactivator of the RAS protooncogene.ne product has an inhibitory effect on RAS, whosehighly expressed in immature and proliferatingack of NF1 inhibitory effect will favor increasedration [46,215]. Initially, it was reported that thereutations of the NF1 gene in bladder cancer; how-studies on the mRNA and protein levels showedin NF1 gene expression in high grade urothelialboth in vivo and in vitro. Therefore, it is believedetic mechanisms may lead to a decrease in NF1ssion in urothelial carcinoma [216].n et al. examined NF1 gene immunohistochemical

in a total of 29 urothelial bladder cancer speci-ced NF1 expression was observed in 23/29 (83%)ecimens and the reduction was more pronouncedde tumors [217].

/LZTS1

zipper putative tumor suppressor 1S1) is a tumor suppressor gene located ate 8p22, which is frequently deleted in humandifferent histotypes. Loss of LZTS1 expressioncantly associated with high tumor grade and atality rate than cancers still expressing the protein

at the short arm of chromosome 8 is a latee development of urothelial carcinoma and iswith invasive disease. A decrease or absence ofin expression is associated with higher grade ofarcinoma [216].

protein is reduced or absent in the majority of blad-cell lines and primary urothelial cancer samples



[218]. Restoration of LZTS1 protein expression in a bladdertumor cell line inhibits cell growth, alters cell cycle pro-gression and suppresses tumorigenicity in nude mice [218].A study inder cancer region of knockout oLZTS1(+/+[219]. In tcarcinoma-bladder canwas absent37/60 prim

5. Growth

A growtof stimulatidifferentiatfactors regsignaling mand hormotarget cellsand matura

5.1. FGFR

Fibroblagene in emproliferatioisoforms, dity cell surFGFR is cof an aminimmunoglofactors (FGand an intrtions are mdominant ssia and hypespecially genetic altelium to papfact that mupapillomaslow grade mutations (nomas. Theextracelluladomain, orhave disclo16% of tumFGFR3 muthat >40% tions in FG

types may indicate that FGFR3 mutations are quite specificfor urothelial tumors.

Mutation in FGFR3 may result in activation of the RAS-K patly a sutions ars. Muinase

low s3 muxima

tions ed to ctic abnmors

eal timxpressoformonly

tion) rs, an3c, aay thint mof low

nt aget founontain

othelia the sus blacy wastions. low tuive re. Eighn, incmutatdifferistry srs comased ent tumage p

of tdvoca/stagein caser canFR3

er canariable

detecd p53molecrade this article in press as: Cheng L, et al. Biomarkers in bladder

cluding 54 bladder tumor samples and 34 blad-cell lines discovered 42% had LOH in the 8p22the tumor samples [219]. Animal models withf LZTS1 gene induced bladder cancer in 8% of), 82% of LZTS1(+/), and 94% of LZTS1(/)he study of FEZ1 expression in five urothelialderived cancer cell lines and 60 primary urothelialcers, Vecchione et al. observed that FEZ1 protein

or significantly reduced in 4/5 cell lines and inary urothelial carcinomas [218].

factors and receptors

h factor is a naturally produced substance capableng cellular metabolism, proliferation, and cellularion. It is usually a protein or a hormone. Growthulate a variety of cellular processes and act asolecules between cells. Examples are cytokines

nes that bind to specific surface receptors of their. Growth factors often promote cell differentiationtion.

3

st growth factor receptor (FGFR) 3 is a crucialbryonic development, cell growth, differentiation,n, and angiogenesis [220]. FGFR has four activeesignated as FGFRs 14. These are high affin-face receptors, encoded on 4p16.3 [221]. Eachomposed of an extracellular domain made upo terminal hydrophilic signal peptide with threebulin-like domains to which fibroblast growthF) bind, a hydrophobic transmembrane domain,acellular tyrosine kinase domain. FGFR3 muta-ost commonly known for causing the autosomalkeletal dysplasia syndromes, such as achondropla-ochondroplasia [220,222]. A mutation in FGFR3,in exons 7, 10, and 15, may be one of the earliestrations in the transformation from normal urothe-illary urothelial carcinoma, as evidenced by thetations in FGFR3 are also identified in urothelial

[223]. Mutations are most commonly found inand low stage tumors, and currently, eight pointmissense) have been identified in urothelial carci-se are usually found to cause substitutions in ther domain, transmembrane domain, cytoplasmic

combinations of these [224227]. Recent studiessed that 74% of pTa tumors, 21% pT1 tumors,ors >pT2, and 0% of in situ carcinomas harbor

tations (Fig. 3) [224,225,228]. It has been reportedof patients with urothelial carcinoma harbor muta-FR3. Lack of this type mutation in other tumor

MAPin onmutatumoPI3 kand FGFRappromutaneedgeneder tu

Rand eent iscomm

liferatumoFGFRpathw

Po88% patiereporalso cin ur

Invarioquenmutawith invasratelyregiowith with chemtumoIncremuta

Stgroupare a

grademon

bladdof FGbladdcal vwere

altereboth low gatol (2013), http://dx.doi.org/10.1016/j.critrevonc.2013.08.008


hway. However, such activation has been foundbset of FGFR3 mutated tumors [229,230]. RASre associated with approximately 15% of bladdertations of PIK3CA, the alpha catalytic subunit of

(PIK3CA), are also associated with low gradetage tumors (but not as strongly associated astations). PIK3CA mutation was demonstrable in

tely 20% of Ta tumors, and coexisted with FGFR3in about 26% of such tumors. More studies arelarify the relationships between FGFR3 and otherormalities in the genesis and progression of blad-

[231].e PCR has been used to detect FGFR3 expressionion of other FGFR proteins in urothelium. Differ-s of FGFR3 have been identified in tumors, most

FGFR3b (which may inhibit FGF-stimulated pro-or FGFR3c (the mesenchymal isoform). In many

isoform switch occurs between FGFR3b andnd this may activate an autocrine or paracrineat stimulates FGFR3 signaling in tumors.utations of the FGFR3 gene were present in up to

grade cancers, but there was no relation with or clinical status [226,232]. Remarkably, oned that approximately 75% of benign papillomased mutations, representing the first genetic defectl papilloma [226].tudy by Tomlinson et al. of 172 patients withdder cancer grades, the FGFR3 mutation fre-

42% [233]. There were 61 single and five doubleThere was a significant association of mutationmor grade and low stage. The noninvasive and

gions from 43 of the tumors were analyzed sepa-teen of these had FGFR3 mutation in at least oneluding nine with mutation in both regions, eightion in only the noninvasive component, and oneent mutations in different regions. Immunohisto-howed overexpression of FGFR3 protein in manypared to normal bladder and ureteric controls.

xpression was associated with mutation (85% ofors showed high level expression).T1 bladder cancer comprises a heterogeneousumors for which different management optionsted. FGFR3 mutation is linked to favorable (low) pTa bladder cancer while altered p53 is com-

es of high grade, muscle invasive (pT2 or greater)cer. van Rhijn et al. investigated the frequencyand TP53 alterations in 132 patients with pT1cer and correlated these data to histopathologi-s and clinical outcomes [234]. FGFR3 mutations

ted in 37/132 (28%) pT1 bladder cancer cases and was seen in 71 (54%). Only 8% of patients hadular alterations. FGFR3 mutation correlated withpT1 bladder cancer and altered TP53 correlated

Please citOncol/Hem


bladde

with high gwere signifimultivariat

TomlinsFGFR3 pr[233]. Muttions, and with low tuoverexpresFGFR3 walium, wherstained negpT1, and 2of FGFR3expressionsive (78% detectable mfactor recepmay repressignaling cmay benefi

MolecuMIB1 exprlogic gradefor Researcmulticenterwith primamutations parameterspT1, high gtion and Manalyses f

ng wa versu

any ctions irecurr

adder f recu

ression evalu

s and FrmatiFig. 3. Key molecular alterations in

rade. FGFR3 mutation status and carcinoma in situcant for predicting progression on univariate and

e analyses.on et al. compared the FGFR3 mutation withotein expression in 158 bladder cancer patientsations comprised 61 single and five double muta-there was a significant association of mutationmor grade and stage. The mutation and protein

sion had an 85% concordance. The expression ofs weak but detectable in normal bladder urothe-eas an increasingly greater proportion of tumors

gradi(89%

Mmutaease

Ta blrisk oprogstudystatuconfoe this article in press as: Cheng L, et al. Biomarkers in bladder canceatol (2013), http://dx.doi.org/10.1016/j.critrevonc.2013.08.008

atively with advancing stage (9% of pTa, 19% of8% of pT2). This suggested that complete loss

expression has biological significance. FGFR3 was higher in noninvasive (57% of pTa) than inva-of pT2) tumors. Overall, 42% of tumors with no

utation still showed overexpression of the growthtor, including many muscle-invasive tumors. This

ent a nonmutant subset of tumors in which FGFR3ontributes to the transformed phenotype and whicht from FGFR-targeted therapies [233].lar grade based on FGFR3 mutation status andession was proposed as an alternative to patho-

in bladder cancer by The European Organizationh and Treatment of Cancer (EORTC) [235]. In a

study, van Rhijn et al. investigated 230 patientsry nonmuscle-invasive bladder cancers. FGFR3were significantly related to favorable disease, whereas altered MIB1 was frequently seen withrade, and high EORTC risk scores. FGFR3 muta-

IB1 labeling index were significant in multivariateor recurrence and progression. The molecular

egorizationMolecular 56% of casOnly 4% oprogressionFGFR3 andindex, 25%and high Kwith a 40%years [235for classifyand permitpatients [7]

The advmay suppoWHO gradAs the mmore sharpscheme, baBecause mlow malignr cancer.

s more reproducible than the pathologic grades 4174%).enters have investigated associations betweenn noninvasive tumors, including FGFR3, with dis-ence and progression. Recent data indicate that fortumors, mutation of FGFR3 confers an increasedrrence but its presence or absence does not predict

in any stage or grade of tumor. A multicenterating bladder tumors for Ki67 immunostainingGFR3 mutation status by PCR with single strand

on polymorphism (PCR-SSCP) resulted in the cat-r: Translational and clinical implications. Crit Rev

of these tumors into three prognostic groups.grade 1 (FGFR3 mutation with a low Ki67 index,es) had a better survival rate than other groups.f molecular grade 1 patients experienced tumor

within 10 years. Molecular grade 2 (wild type low Ki67 index or mutant FGFR3 and high Ki67

of cases) or molecular grade 3 (wild type FGFR3i67 index, 19% of cases) were together associated

risk of patients showing tumor progression at 10240]. These studies allude to a future potentialing bladder tumors based on molecular makeupting less stringent followup of molecular group 1.

ances in molecular genetics of bladder neoplasmsrt modification to the currently proposed 2004ing system for bladder neoplasms [241243].

olecular profiles of the various entities comely into focus, perhaps a separate classificationsed on these findings, will be eventually used.

any papillomas, papillary urothelial neoplasms ofant potential, and low grade papillary urothelial



carcinomas show similar genetic profiles, this may eventu-ally change the currently established definitions and onceagain reorganize the process of bladder tumor diagnosis[241,242]. pressor genwell [7,16,

5.2. EGFR

Epidermkinase, simlocated ongrowth-regligand bindtor (EGF) Binding ofof the recepathways [245,246]. role in prois identifieoverexpresoverexpresaggressive [247,248]. expressed i[249]. Currnonurotheltherapy. Mthe extracesine kinasesynergistictance to thecancers, an

synthesis athose cancOverexpresamplified iinvasive tu

EGFR including tEGFR immder cancers(71% of thbranous aninvasive cawith bladdvival [256,found progfactor TGFassociationeration indcancer sugpromotion tion of incgroup antig

defective glycosylation is involved in urothelial carcinogene-sis and progression [259]. EGFR immunoreactivity may berelated to expression of JUN oncoprotein, which interacts

over 3e levr tum

e progion o

pT1val [2ion wR expfic moval inctomy

or mo

nts wihaux ssion

y PCRfin-emivity w

cases

result tationithelicated NA)

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re EGts of

R-indeherin

expreigher globival [2

VEGF

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or grapears tDownthe swe, wh.

EGF inumbtumor this article in press as: Cheng L, et al. Biomarkers in bladder

Establishing the presence of LOH in tumor sup-es may play a major role in these definitions as

167,244].

/ERBB1

al growth factor receptor (EGFR) is a tyrosineilar to FGFR3. It is encoded by a protooncogene

chromosome 7p13. EGFR is a transmembraneulating 170 kd glycoprotein. The extracellularing site is a receptor for epidermal growth fac-and transforming growth factor alpha (TGFA).

EGF to its receptor results in downregulationptor and stimulation of tyrosine kinase signalingfor cell migration, adhesion, and proliferationOverexpression of EGFR plays an importantmoting carcinogenesis. Amplification of EGFRd in 5% of urothelial carcinomas and proteinsion is found in 23% of bladder tumors. EGFRsion is associated with poorer prognosis and moretumor behavior than other low grade tumorsSome studies have indicated that EGFR is over-n as many as 3050% of invasive bladder cancersently, Phase 1 trials are underway in patients withial solid tumors, exploiting two sites for targetedonoclonal antibodies specifically directed againstllular domain of EGFR, and blockade of the tyro-

intracellular domain of the receptor may operateally to reduce the probability of acquired resis-rapy. Gefitinib has been recently studied in lung

d the resulting studies indicate that it inhibits DNAnd decreases tumor cell numbers, especially iners that harbor mutations in EGFR1 [250252].sion of EGFR and ERBB2, a gene frequentlyn breast cancer, are frequently associated withmors that carry a worse prognosis [253,254].expression can be seen in normal conditions,he basal cell layer of normal urothelium [255].unoreactivity is present in about 50% of blad-

, with increased expression in stage T24 cancersese cases) [256]. Staining is predominantly mem-d most prominent at the advancing edge of thencer [256]. EGFR immunoreactivity correlates

er cancer recurrence, time to recurrence, and sur-257]. One study of 43 invasive tumor patientsnostic va

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Biomarkers in bladder cancer: Translational and clinical implications