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Molecular Markers for Diagnosis, Staging, and Prognosis of Bladder Cancer

Molecular Markers for Diagnosis, Staging, and Prognosis of Bladder Cancer

ABSTRACT: Conventional histopathologic evaluation of bladder cancer, encompassing tumor grade and stage, is inadequate to accurately predict the behavior of most bladder tumors. Intense research efforts are under way to identify and characterize various bladder cancers and their true biological potential more effectively. The need to predict which superficial tumors will recur or progress—and which invasive tumors will metastasize—has led to the identification of a variety of potential prognostic markers for patients with bladder cancer. The molecular changes that occur in transitional cell carcinoma of the bladder are numerous and can be categorized into (1) chromosomal alterations, leading to carcinogenesis; (2) loss of cell-cycle regulation, accounting for cellular proliferation; and (3) growth control events such as angiogenesis, resulting in metastasis. It is becoming apparent that the accumulation of genetic and molecular changes ultimately determines a tumor’s phenotype and subsequent clinical behavior. The potential clinical application of new diagnostic techniques (ie, loss-of-heterozygosity analysis to identify tumor suppressor genes) and older, well-established, techniques (ie, immunohistochemistry) combined with improvements in the use of automated and standardized systems are areas of active investigation. [ONCOLOGY 15:1461-1484, 2001]

Transitional cell carcinoma of the bladder, the second
most common tumor of the genitourinary tract, is also the second most common
cause of death from these cancers. In 2001, an estimated 54,300 new cases will
be diagnosed in the United States, and some 12,400 patients will die of the

Approximately 80% of patients with primary bladder cancer experience
low-grade tumors confined to the superficial mucosa, and the majority are
amenable to initial transurethral resection and selected administration of
intravesical immunotherapy or chemotherapy.[2] The risk of recurrence for
patients with superficial bladder tumors can be as high as 70%, however.
One-third of recurrent tumors may demonstrate tumor progression to a higher
grade and/or stage of disease. Muscle-invasive tumors occur initially in 15% to
30% of all bladder cancer patients; 50% of those treated locally for invasive
tumors will relapse with metastatic disease within 2 years of treatment.[3]
These data underscore the heterogeneous nature and malignant potential of
transitional cell carcinoma of the bladder.

The optimal management of invasive bladder cancer requires the detection and
accurate assessment of the tumor’s biological potential. Treatment strategies
for patients with bladder cancer are currently dictated by histologic
evaluation, including determination of tumor grade and stage as the primary
prognostic variables. Although these two conventional histopathologic variables
provide a certain degree of stratification of a tumor’s biological
characteristics, there remains a significant degree of tumor heterogeneity
within the various prognostic subgroups. This makes it difficult to accurately
and reliably predict the tumor’s aggressiveness. The ability to precisely
predict an individual tumor’s true biological potential would facilitate
treatment selection for patients who may benefit from adjuvant therapy and
identify patients who may require less aggressive strategies. Intense research
efforts are ongoing to best characterize bladder cancer and its varying
biological profiles.

Transitional cell carcinoma of the bladder has generally been viewed as two
different disease processes. Superficial tumors are most often locally
proliferative and recurrent but can become invasive and even metastatic.
Superficial tumors that maintain a malignant phenotype may be treated more
effectively with early, aggressive intravesical therapy or even cystectomy. The
use of molecular markers may guide decision-making in the treatment of
superficial bladder cancers.[4] Muscle-invasive cancer, notorious for its
potential clinical virulence, is ideally treated aggressively,[2] but there
remains a significant incidence of recurrence and disease progression in some
patients, who may ultimately benefit from an adjuvant form of therapy.

The need to predict which superficial tumors will recur or progress—and
which invasive tumors will metastasize—has led to an ongoing search for
improved understanding of bladder carcinogenesis and metastasis. With the advent
of new molecular techniques, the field of medical molecular biology has exploded
in recent years, resulting in detailed analysis of human cells and tissues on
the DNA, RNA, and protein levels. The molecular and genetic changes in
transitional cell carcinoma of the bladder can be schematically classified into
three separate, but intertwined, events: (1) chromosomal alterations,
representing the initial event in carcinogenesis; (2) tumor proliferation,
caused by loss of cell-cycle regulation; and (3) metastasis, in which the
initial tumor breaks from its original confined environment, aided, in part, by
processes such as angiogenesis and loss of cell adhesion.

We believe that the accumulation of these successive genetic alterations—rather
than a single genetic event in time—determines a tumor’s phenotype and,
subsequently, the patient’s clinical outcome. In this review, we will
summarize the recent literature regarding molecular and genetic changes in
bladder cancer and comment on potentially improved diagnostic tools and
treatment regimens becoming available as a result of our better understanding of
these molecular pathways.

Expanding Diagnostic Modalities

Our understanding of tumor biology has evolved rapidly over the past decade.
Advances in molecular biology, immunology, and cytogenetics have prompted this
progress. An increase in knowledge has provided an opportunity to identify and
evaluate tumor characteristics beyond the scope of general histology and gross
DNA content and to distinguish the potential behavior of an individual tumor. As
the role of tumor markers in the diagnosis and prognosis of bladder cancer
grows, it is important to understand the various technologic, methodologic, and
analytic issues regarding each diagnostic modality. Several techniques can be
applied to evaluate a tumor marker; most are only applied in a research setting,

For the clinical urologist, the most widely used evaluation method is
immunohistochemistry. Translational research has enhanced the application and
evaluation of immunohistochemical techniques with potentially important clinical
roles in bladder cancer. Currently, most tumor markers that have been studied
and merit a role in the contemporary clinical decision-making process for
bladder cancer have evolved from the application of immunohistochemistry.

Standardization of the technique and its interpretation will ultimately be
necessary for successful and consistent application of immunohistochemistry.
With proper controls and standardization, immunohistochemistry will remain one
of the principle techniques used to evaluate various tumor markers. Although
efforts have been made to standardize immunohistochemistry, the limitations of
this technique must be realized before we can develop a consensus as to how to
perform the procedure. Despite these limitations, immunohistochemistry maintains
some translational application in the analysis of various bladder cancer
markers. Other more sophisticated and costly techniques, such as single-strand
conformational polymorphism, DNA sequencing, or polymerase chain reaction (PCR)-based
analyses, are currently used in the research setting but are not yet applicable
to clinical decision-making.

Carcinogenesis of Bladder Tumors


Bladder cancer is an excellent model for the study of molecular changes at
the DNA level, owing to its distinctly different subtypes (superficial and
muscle-invasive) and their different propensities to progress. Such DNA
alterations in bladder cancer have been studied in a variety of ways, ranging
from cytogenetics to DNA ploidy to loss of heterozygosity.[5] DNA alterations
can result from a number of genetic insults such as point and insertional/deletional
mutations, translocations, and loss of alleles. Each insult may affect the
translated protein product. The large fund of molecular knowledge developed in
recent years about carcinogenesis has provided some evidence about the different
genetic pathways for bladder cancer.

Earlier work in the field of molecular oncology focused on oncogenes, which
are normal cellular genes that contribute to the malignant phenotype of a tumor
by overexpressing the normal gene product or, in some cases, by expressing a
protein product with altered function. Overexpression of the normal gene product
is usually achieved by gene amplification or chromosomal translocation of the
gene to an area downstream of a powerful promoter. However, expression of a
mutated protein product can also lead to activation of the malignant phenotype.
Oncogenes believed to be important in human malignancies include cH-ras, c-myc,
MDM2, and HER2/neu (aka c-erbB2).

cH-ras Gene—The cH-ras gene is an active oncogene thought to be involved
in the development and progression of human bladder cancer. Mutational studies
of the ras gene family have demonstrated that alterations in codons 12 and 61 of
the H-ras gene occur in up to 20% of bladder cancers.[6-8] One study, employing
PCR amplification followed by oligonucleotide-specific hybridization, reported
that 36% of bladder tumors had the same mutation on codon 12 of the H-ras
gene.[9] In general, the activation of H-ras occurs by a single-point mutation
(G d A) in codon 12, although other mutations have been described.[6]

Clinically, Fontana and colleagues demonstrated a statistically significant
relationship between the overexpression of the c-ras oncogene and early
recurrence in patients with superficial bladder cancer.[10] These data suggest a
potential prognostic role for the c-ras oncogene in patients with superficial
bladder cancer, but currently these techniques apply only in a research setting.

c-myc Gene—An important regulator of cellular proliferation, the
gene family encodes for nuclear phosphoproteins containing DNA-binding
activity.[11] The c-myc oncogene has been shown to be overexpressed in several
human tumors including bladder cancer.[12,13] Deregulation of the myc gene
family occurs with chromosomal translocation and gene amplification,[14] and
studies have demonstrated that myc overexpression promotes cellular
proliferation.[10] Although the genetic mechanism causing overexpression of the
c-myc gene in bladder cancer is unknown, its overexpression has been shown to be
associated with high-grade bladder cancer.

Kotake and associates demonstrated that expression of the c-myc gene product
correlates with the nuclear grade of bladder cancer.[13] In a study with
conflicting results, however, Lipponen found no independent prognostic value for
Myc proteins with respect to prognosis for patients with transitional cell
carcinoma of the bladder.[15] Currently, the prognostic significance of c-myc
gene expression is unknown, and further evaluation will be required to determine
its prognostic role.

HER2/neu—The proto-oncogene HER2/neu has been extensively studied and
implicated in a number of tumors, including breast, prostate, and bladder
cancers.[16] The HER2/neu oncogene encodes a transmembrane glycoprotein similar
to the epidermal growth factor (EGF) receptor, having tyrosine kinase
activity[17] and the ability to stimulate cellular growth.[18]

Initial studies of HER2/neu were performed in breast carcinoma and
demonstrated a significant relationship between gene expression, tumor
progression, and overall survival.[19] Subsequently, several studies reported
that HER2/neu expression in bladder cancer patients is associated with
higher-stage tumors,[20-22] increased tumor progression,[16] greater incidence
of metastasis,[22] and reduced overall survival.[20] These investigations
suggest a prognostic value of HER2/neu expression in human bladder cancer; other
studies have reported conflicting results, concluding that evaluation of the
oncogene provides no additional prognostic value over the previously established
predictors of grade and stage for transitional cell carcinoma.[23,24]

In light of these discrepant results, further evaluation will be required to
accurately determine the prognostic value of HER2/neu in bladder cancer.


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