Bladder Cancer (BC) is an increasingly important international public health problem. As a multifactorial disease, both environmental and genetic factors are involved in its development and progression for bladder cancer. Risk factors for BC include older age, male sex, white race, smoking, occupational exposures, infections caused by certain parasites and a family or personal history of bladder cancer. Factors that influence risk of progression include the tumor stage and grade, the number, size, the response to initial treatment and others. Although, various environmental factors contributing to BC pathogenesis have been studied, genetic variations due to ethnic differences, which may play a major role in its susceptibility in India, are relatively unexplored. Moreover, the current understanding suggests that genetic factors and their interaction with the environment may provide a relatively better picture of the factors involved in the disease pathogenesis. Because of the lifetime need for surveillance, the treatment of recurrent tumors and the cost of complications associated with treatment, BC poses a significant economic burden. A major challenge for screening is to accurately identify tumors that are still superficial, yet at high risk for progression, in order to initiate interventions at an early stage.
Intravesical Bacillus Calmette-Guerin (BCG) immunotherapy is effectively being used to reduce recurrence and progression in non-muscle-invasive bladder cancer but the dosing schedule and duration of treatment remain empirical. There is thus, increasingly good evidence that BCG maintenance therapy, at the optimal treatment schedule, provides greater protection from tumor progression and recurrence, and improves long-term survival (Lamm, 2006). Several effects, ranging from a direct antiproliferative/cytotoxic effect of BCG, to a role in the initiation and/or modulation of the host immune response, and to an increase of susceptibility of tumor cells, have been proposed (Bevers et al., 2004).
Among the most common molecular changes present in tumors including BC are mutations of House-keeping genes, angiogenesis and loss of cell-cycle regulation. Microsatellite instability and loss of heterozygosity are hallmarks of various cancers and these genetic changes have also been found at several loci in BC. Therefore, recent molecular epidemiological studies have been shifting towards a more comprehensive pathway based approach to assess the complex interactions between multiple genes and environmental exposure and to make a more robust assessment of risk groups. Perera and Weinstein (1982) have addressed “molecular epidemiology” as new approach which especially considers association of genetic factors. In addition to environmental components in multifactorial diseases like cancer, association studies may be family based or population based (Perera and Weinstein ,1982). Population based case control studies employ unrelated cases and control subjects for exploring the significant difference of allele/genotype frequencies in cases and controls as an estimate of risk for developing particular disease.
Polymorphic variations in humans may be responsible for inter-individual differences in susceptibility to multifactorial diseases. Thus, association studies provide statistical power to reveal the contribution by risk alleles. The goal of a genetic association analysis is to reveal the contribution by risk alleles and to show that a disease phenotype varies in a predicable manner with a marker genotype. Significant differences in allele frequencies between cases and controls are taken as evidence for involvement of an allele in disease susceptibility. Case-control analysis is thus a well validated technique for the discovery of alleles associated with human disease susceptibility. Case control designs suffer due to low response rate of environment exposure (like smoking and use of alcohol) in population controls, resulting in non-comparability of cases and controls (Wacholder et al., 1992; Vineis et al., 2004). In such situations, case only designs have been suggested as superior alternative.
The recent shift to an agnostic genome-wide association approach led to the identification of several BC susceptibility loci, and provided valuable leads for new mechanistic insights into BC carcinogenesis. The markers do not have sufficient discriminatory ability yet to be applied for risk assessment in the population and the question is whether they ever will. Prognostic and predictive studies in BC are still in their infancy compared with etiologic studies.
SNPs in the coding region of gene may lead to an amino acid change that may alter protein function. Regardless, given their potential role on expression and phenotype, polymorphisms may account for the differences seen in susceptibility to and the outcome from a given disease (Lohmueller et al., 2003). However, choosing an ideal control set for a population study is a difficult problem, as the age, sex and ethnicity of the case and control groups should be matched to enable appropriate conclusions to be made. Studies on functional gene polymorphisms are more likely to yield positive results than random polymorphisms simply because of the greater prior probability of being associated with disease. However, other gene variants, even if not functional, can be associated with phenotype because of linkage to closely situated functional polymorphisms. It is now recognized that specific combinations of polymorphisms in a gene (i.e. Haplotypes) might be of greater significance than individual polymorphisms, not only for a more efficient capture and analysis of common genetic variation (Johnson et al., 2001).
Cancer stem cells (CSCs), are a population of cells present in tumors, which can undergo self-renewal, differentiation and can also give rise to cancer cells in a tumor (Tanabe, 2010). Due to these properties of CSCs they can be targeted for cancer prevention and treatments. Further understanding of the distinction between normal stem cells and cancer stem cells can lead to the development of effective regimen that can either be used to eliminate the stemness of cancer cells making them susceptible to standard therapies or eliminate the CSCs themselves (Madka & Rao, 2011). Highly malignant tumors mostly consist of rapidly proliferating cells. However, tumors also contain a few cells in a quiescent state that can be characterized as slow-cycling, expressing markers of stem cells and possessing the ability to initiate new tumors. These quiescent cells, generally termed ‘cancer stem cells’ (CSC) (or ‘cancer initiating cells’), are capable of regenerating the entire tumor—as it occurs in metastatic spread. Although each type of cancer may have CSC markers that are specific to that tumor (for example, CD271, in melanoma stem cells). While these markers and variants of them are highly relevant for studies on the biology of cancer. Some prominent among these are CD24, CD26, CD28, CD166, CD44, EpCAM, and ALDH.
CD44 (Cluster of differentiation 44): CD44 is a multifunctional class I transmembrane glycoprotein (Naor et. al., 2008) generally acts as a specific receptor for hyaluronic acid, promoting migration in normal cells and highly expressed in almost every cancer cell in its standard or variant form (Ponta et. al., 2003). It is mainly associated with proteins that monitor the extracellular changes and critical in regulating cell adhesion, proliferation, growth, survival, motility, migration, angiogenesis, and differentiation (Du et. al., 2008, Naor et. al., 2008). Also, CD44 presents cytokines and chemokines to their complimentary receptors on the cellular membrane (Naor et. al., 2008). CD44 interacts with osteopontin and regulates its cellular functions leading to tumour progression. CD44 is expressed on cancer cell surface and assist haematogenous spread while interacting with P- or L-selectins (Napier et. al., 2006). It is also involved in numerous complex signalling cascades enhancing tumour initiations by interacting with neighbouring receptors like tyrosine receptor (Jaggupilli et. al., 2012). Winder et. al., in univariate analysis found CD44 rs187115 and CD44 rs755392 are significantly associated with tumor recurrence and survival in gastric adenocarcinoma patients (Winder et. al, 2011)
CD24 (Cluster of Differentiation 24): CD24 is a small cell surface protein molecule anchored by glycosyl-phosphotidyl-inositol in a wide variety of cancer cells. It is heavily glycosylated and functions in cell-cell and cell-matrix interactions. CD24 is highly expressed in ovarian, breast, prostate, bladder, renal, non-small cell carcinomas, and other human cancers (Kristiansen et. al., 2003, Zheng et. al.,2011). It is involved in cell adhesion and metastasis (Lee et. al., 2010). This indicates that CD24 could be a significant marker in tumour prognosis and diagnosis. Functionally, it is identified as an alternate ligand for P-selectin, an adhesion receptor on platelets and endothelial cells (Aigner et. al., 1998), through which their interaction facilitates the passage of tumour cells in blood stream during metastasis. It increases proliferation and adhesion of tumour cells to fibronectin, collagen, and lamin (Zheng et. al., 2011). The metastatic associations of CD24 increase its importance as a prognostic factor and a new CSC marker (Jaggupilli et. al., 2012). Zhou et. al. 2003 found 2 fold increased risk among the individuals havind CD24 SNP, the study also suggests that the CD24 gene may be a modifier for Multiple Sclerosis susceptibility (Zhou et. al., 2003).
CD26 (Cluster of differentiation 26): CD26 is a cell surface glycoprotein receptor involved in the costimulatory signal essential for T-cell receptor (TCR)-mediated T-cell activation. It acts as a positive regulator of T-cell coactivation, by binding at least ADA, CAV1, IGF2R, and PTPRC. It’s binding to CAV1 and CARD11 induces T-cell proliferation and NF-kappa-B activation in a T-cell receptor/CD3-dependent manner. Its interaction with ADA also regulates lymphocyte-epithelial cell adhesion. In association with FAP is involved in the peri cellular proteolysis of the extracellular matrix (ECM), the migration and invasion of endothelial cells into the ECM. It may be involved in the promotion of lymphatic endothelial cells adhesion, migration and tube formation when overexpressed, enhanced cell proliferation, a process inhibited by GPC3. It acts also as a serine exopeptidase with a dipeptidyl peptidase activity that regulates various physiological processes by cleaving peptides in the circulation, including many chemokines, mitogenic growth factors, neuropeptides and peptide hormones removes N-terminal dipeptides sequentially from polypeptides having unsubstituted N-termini provided that the penultimate residue is praline (De Chiara et. al., 2010). Higashibata et. al. 2013 showed that the. Haplotype analysis C-A (rs7608798- rs2268889) haplotype was significantly associated with increased PSA levels, compared with the most common haplotype, T-A. The study also suggested that DPP4 genetic variants influence baseline PSA levels, especially in men aged between 60 and 69 (Higashibata et. al. 2013).
CD28 (Cluster of differentiation 28):CD28 is a well expressed antigen on human T- lymphocyte, plays an essential role in T- cell activation and T-cell-B- cell interaction through its specific ligands. Also the signaling through T- cell receptor is necessary to induce antigen specific T- cell activation and cytokine secretion. A co-stimulatory signal involving the T- cell surface molecule CD28 and its ligand is essential for T-cell clonal expansion (Gruber et. al., 2008). The protein encoded by this gene is essential for T-cell proliferation and survival, cytokine production and Th-2 type development. Functionally, it is involved in T- cell activation, the induction of cell proliferation and cytokine production and promotion of T- cell survival (Hashemi et. al., 2010). Hashemi et. al. 2010 in one of their findings suggest that the CD28 gene may have significant role in the protection of active visceral leishmaniasis (VL) in the Iranian population.
CD166 (Activated Leukocyte Cell Adhesion Molecule (ALCAM)): Activated leucocyte cell adhesion molecule (ALCAM; CD166) is a cell adhesion molecule that belongs to the immunoglobulin superfamily and is widely expressed in a variety of normal tissues (Weichert et. al., 2004). It functions as a ligand to CD6 and also mediates homophilic (ALCAM–ALCAM) interactions. This gene encodes activated leukocyte cell adhesion molecule (ALCAM), also known as CD166 (cluster of differentiation 166), which is a member of a subfamily of immunoglobulin receptors with five immunoglobulin-like domains (VVC2C2C2) in the extracellular domain. This protein binds to T-cell differentiation antigen CD6, and is implicated in the processes of cell adhesion and migration. Due to this property of the molecule it helps in cancer progression and also tumorigenesis (Weichert et. al., 2004). Weichart et. al. has reported that ALCAM is upregulated in case of colorectal cancer and the membranous expression of ALCAM has significantly decreased the survival of patients with colorectal cancer. (Weichert et. al., 2004).
Smoking is the most significant risk factor for BC susceptibility, with smokers having three times the risk of bladder cancer compared with nonsmokers (Zeegers et al., 2000). This risk is greatest for patients with advanced stage, invasive disease (Sturgeon et al., 1994). Despite this, not all smokers go on to develop BC. Therefore, there may be genetic factors that predispose some smokers to be at great risk for the development of invasive bladder cancer.
The specific aim in cancer research is to identify the causative genes and mutations leading to carcinogenesis. This knowledge can then be translated into new targets for diagnosis and treatment. The continuing investigation into the genetic basis of cancer has revealed a number of genes whose individual or concerted actions, when mutated, results in oncogenesis. So far, there are few published reports on genetic susceptibility to BC in North Indian population. Identification of genetic factors associated with bladder cancer may be useful in disease management and therapy. Therefore, the present case-control study was undertaken to investigate the association of CD44, CD166, CD24, CD26 and CD28 gene polymorphisms with bladder cancer susceptibility in north Indian population. The genetic variants were detected in the well-defined selected north Indian population by using PCR-RFLP and TaqMan allelic discrimination assay methods depending upon the type of sequence variants which might be helpful in better understanding of the disease pathogenesis. The association of genotypes with tumor grade and stage of the tumors in patients was also determined. A case only analysis was carried out to explore the modulation of BC risk due to smoking and its correlation with genotypes.
The effect of adjuvant BCG therapy on patients with recurrence and without recurrence may provide a deeper understanding on the pathophysiology of the disease. Multiple studies have demonstrated that transurethral resection of transitional cell carcinoma (major form of bladder cancer) followed by intravesical instillation of BCG in the treatment of and prophylaxis against recurrent stages Ta and T1 tumors and carcinoma in situ significantly reduces recurrence and prolongs disease free survival (Mignon et al., 2002). Kaplan-Meier analysis was performed to analyze the association of these polymorphisms with disease free recurrence and survival in BCG immunotherapy treated patients. Classification And Regression Tree (CART) and Multifactor Dimensionality Reduction (MDR) approaches were combined with logistic regression to discover high order gene-gene interactions and for overcoming the limitations of logistic regression, sample size for present study.
The results of the study may significantly add to our understanding of genetic factors allied with bladder cancer development and ascertain some light on the role of disease in BC pathogenesis. In future, focus on a genome-wide association approach possibly using whole-genome sequence data, consortia formation and meta-analyses, and blood and tumor tissue collection, preferably in the context of randomized controlled trials will stimulate well designed and sufficiently powered studies, and thereby enhance the elucidation of genetic prognostic and predictive markers.