Evolving Paradigms In Bladder Cancer: Emerging Biomarkers

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Many candidate biomarkers are originally detected by immunohistochemistry (IHC). Other methods for biomarker detection include enzyme-linked immunosorbent assay (ELISA), real-time polymerase chain reaction (RT-PCR), FISH, and more specialized assays.35,36 A select group of potential biomarkers being investigated for clinical application in bladder cancer:

• Urothelial carcinoma demonstrating absence of PD-L1 staining (20×).
• Urothelial carcinoma demonstrating marked membranous PD-L1 staining (20×).

Programmed death ligand 1 (PD-L1) is a coinhibitory repressor that negatively regulates T-cell responses. It is highly expressed in several cancers, including bladder cancer, and has been associated with localized stage advancement of urothelial carcinoma and attenuation of responses to BCG therapy.^36-40Based on reports from a phase I study with the anti—PD-L1 antibody atezolizumab (MPDL3280A)⁴¹in patients with metastatic bladder cancer demonstrating tumor shrinkage in 43% of patients with PD-L1-positive metastatic urothelial bladder cancer, the agent received a breakthrough therapy designation by the FDA.⁴²Recently updated results from the phase II IMvigor210 trial⁴³of atezolizumab in heavily pretreated patients with locally advanced or metastatic urothelial carcinoma reported a promising median overall survival (OS) of 7.9 months in the overall study population, and 11.9 months among patients whose tumors expressed medium and high levels of PDL1.⁴⁴Testing for PD-L1 status is likely to be beneficial in determining patients who may benefit from anti—PD-L1 therapy.

BLCA-4 is a nuclear matrix protein transcription factor that has been identified as an abundant bladder cancer-specific protein. Its overexpression has been determined to induce cell proliferation and the production of mediators involved in invasion and angiogenesis.⁴⁵An investigational ELISA assay using urine samples is in development for BLCA-4 detection in individuals with a high risk for bladder cancer.¹⁶

Telomerase is an enzyme that maintains chromosomal integrity by preserving telomeric DNA that would otherwise be degraded during rapid cell division of cancer cells.⁴⁶This enzyme is present in more than 80% of all cancer cells while not expressed in most healthy cells, indicating its potential for success as a bladder cancer biomarker. Telomerase reverse transcriptase mRNA is detected in exfoliated cells in urine by telomerase reverse transcriptase assays or a PCR-based telomeric repeat amplification protocol (TRAP).16 Telomerase has also been detected using IHC and has been found to independently associate with reduced recurrence-free survival and the development of invasive disease in patients treated with BCG therapy following TURBT.⁴⁷Thus, telomerase expression may be useful for identifying patients who are at higher risk for recurrence and who might benefit from radical cystectomy.

Fibronectin is a glycoprotein that is expressed in cells, plasma, and the ECM and has been reported to be associated with bladder cancer. Bladder tumor fibronectin (BTF) is an assay that is being developed to measure urinary fibronectin for the detection of bladder cancer. One study has shown that BTF predicts residual tumor load following TURBT with high sensitivity (91.4%) and specificity (87.8%) in patients with suspected bladder cancer. Further investigation is under way to develop this assay and gain FDA approval.¹⁶

Survivin is a protein that inhibits apoptosis by blocking caspase activation. It has been detected by mRNA and protein in urine samples nearly exclusively in patients with bladder cancer compared with healthy individuals and has also been shown to be expressed by malignant epithelium.⁴⁸Survivin has been reported as superior to NMP22 and cytology for bladder cancer detection, demonstrating better sensitivity, specificity, and both positive and negative predictive values.⁴⁹However, a drawback of survivin measurements in the urine is the release of survivin by the healthy prostate.⁵⁰Measuring the proportion of survivin that originates from the prostate may be challenging in men of varying prostate sizes.

FGFR3 is a fibroblast growth factor receptor (FGFR) that is commonly mutated or overexpressed in bladder cancer, resulting in increased cell proliferation. FGFR3 mutation is strongly associated with low-grade and low-stage bladder tumors and is linked to a favorable disease course with low features of clinical malignancy. Overexpression of wild-type FGFR3, however, is associated with invasive bladder cancers and activates Ras/ mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) pathways, which are involved in cell proliferation and survival. FGFR3 can be detected in urine and is currently being developed into an assay for bladder cancer detection and prognosis.⁵¹

Matrix metalloproteinases (MMPs) are ECM degradation enzymes that play important roles in tumor cell invasion and metastasis. MMP2 has been studied for its role in invasive urothelial carcinoma due to its ability to degrade components of the bladder’s ECM, including type IV collagen, gelatin, and laminin. High levels of MMP expression measured by RT-PCR correlated with worse survival in patients with urothelial carcinoma.⁵²Another study measured MMP2 protein expression by ELISA in serum samples in patients with urothelial carcinoma and determined that MMP2 expression was predictive of recurrence and advanced disease.⁵³Currently, MMP2 measurement in urine is being developed as a marker of disease prognosis.²⁵

This list of candidate biomarkers is by no means exhaustive. Several other molecular markers are also under investigation for bladder cancer, including hyaluronic acid, DNA methylation markers, gene expression signatures, cytokeratins, prothymosin alpha, soluble Fas, Reg1, p53, Rb, Ki67, and more.^16,25,35