Single Nucleotide Polymorphism in PSCA Associated with Bladder Cancer Susceptibility in an Indian Subpopulation

doi:10.21203/rs.3.rs-4142409/v1

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Single Nucleotide Polymorphism in PSCA Associated with Bladder Cancer Susceptibility in an Indian Subpopulation

https://doi.org/10.21203/rs.3.rs-4142409/v1

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Background

Urothelial bladder cancer(UBC) is the most common neoplasm of the urinary system. Genome Wide Association Studies (GWAS) have reported that single nucleotide polymorphisms in the PSCA gene are associated with BC risk. Prostate stem cell antigen genes play a role in cell proliferation inhibition and cell death induction. The expression of PSCA is altered in BC. It may be a useful marker for diagnosis and disease progression of UBC.

Materials and methods

In this hospital-based study, we evaluated the risk factors for bladder cancer and their associations with single nucleotide polymorphisms in the PSCA gene. rs2294008C/T genotyping was performed by real-time Taqman® probes in histologically confirmed BC patients (107) and healthy controls (105) from a tertiary care hospital. Statistical analysis of association studies was performed with SPSS ver 22.0.

Results

The odds ratio for heterozygosity for CT and variant allele T of rs2294008 were 1.71 and 1.82 respectively. Thus there is an increased risk of BC due to polymorphisms. When the PSCA rs2294008C/T heterozygous CT genotype for high-grade tumors was combined with the tumor-grade, a substantial BC risk was found (p = 0.001;OR = 1.984). For individuals with the PSCA rs2294008C/T gene polymorphism heterozygous for the CT genotype (p = 0.0001), smoking significantly reduced the incidence of BC.

Conclusions

Our research revealed that a complicated intervention involving PSCA rs2294008C/T confers a greater risk of BC risk in the North Karnataka population

Prostate stem cell antigen

Bladder cancer

Single nucleotide polymorphism

Genome-wide association study

According to the GLOBOCAN registry 2020, there have been almost 2 million cases of bladder cancer reported in Asia, with India accounting for 10% of the cases and ranking third overall. Bladder cancer (BC was the tenth most common cancer globally in 2018. Seventy-five percent of BC patients have non-muscle-invasive BC (NMIBC), and the majority of cases are urothelial carcinoma (UC) [1]. Because of its high recurrence rate and slow natural history, NMIBC is very common. The most typical signs of BC patients are hematuria or lower urinary tract symptoms, while other symptoms can be silent or unexpected [2]. External risk factors are linked to the majority of BC cases and aluminum production, and include exposure to different dyes such as magenta and auramine or dye intermediates such as 4-aminobiphenyl), environmental exposures (such as gamma or X radiation, and arsenic), medications (such as cyclophosphamide and chlornaphazine), opium intake, and Schistosoma infection [3–5]. These findings effectively broaden our understanding of the mechanisms underlying carcinogenesis [6]. Expanding our understanding of the fundamental pathophysiology of malignancies at the gene level is crucial, especially in light of the practical importance of genetic variants in cancer inititation and development. Genetic differences that biologically regulate a few molecular processes, such as sequence changes and abnormal cellular genome structure including single-nucleotide substitutions to large chromosomes, cause cancer formation. The prostate stem cell antigen(PSCA) gene is located on chromosome 8q24.2 [7, 8]. PSCA is a glycoprotein cell surface marker that is overexpressed in prostate cancer cell lines [9].¹⁰ The regulation of PSCA varies in different types of cancers. Various studies have reported that the PSCA is downregulated in esophageal, gastric and gallbladder carcinoma whereas the PSCA is upregulated in solid tumours like pancreatic, bladder, renal cell carcinoma, and ovarian mucinous [10, 11]. A comprehensive meta-analysis showed that rs2294008 polymorphism was a moderate risk factor for bladder cancer in Caucasian and Asian populations [11]. Significant effects on changes in the transcriptional activity of an upstream PSCA fragment may be attributed to rs2294008 in the first exon of the PSCA gene [12]. The evidence supporting the function of the PSCA rs2294008 polymorphism as a marker of genetic origin for cancer risk is still equivocal, even though mounting data have shown a correlation between the polymorphism and cancer risk [13, 14]. GWAS data on the Indian population are sparse. Therefore, in this study we explored the association between the PSCA rs2294008 polymorphism and cancer risk in a group of South Indians.

2.1. Study subjects: All enrolled patients had incident cases of histologically confirmed UBC and were recruited from the Urology Department at KLES Dr Prabhakar Kore Hospital and Medical Research Centre. A total of 107 patients with histologically confirmed BC (mean age 59.8 years; 84 males and 23 females) were recruited. Patients with a history of other cancers, who had previously undergone radiation therapy, or who had metastatic cancer of another origin were excluded. Age- and sex-matched controls without any genetic history of cancer, or chronic disease were included. (n = 105). The mean age of the controls was 58.9 years. To gather information on the research participants' demographics, smoking and employment histories, and other lifestyle variables, a questionnaire was created. A 4 mL blood sample was taken into a coded EDTA tube at the conclusion of the interview. All the subjects provided their informed consent. The study was authorized by the KAHER Institute's Ethical Review Board.

2.2. Epidemiology data collection: Each person in the case and control group was interviewed to obtain demographic information. Individuals who smoked once a day for more than 5 years were defined as smokers, similar to alcohol and tobacco consumption. Individuals who had never smoked or been exposed to other habits in their lifetime were regarded as nonsmokers (similar definitions for other exposures). After the interview, a 4 ml blood sample was drawn into EDTA vials [15]. ¹⁸ Of the 107 total patients enrolled in the study, 39 (36.4%) patients had non–muscle-invasive bladder

2.3. Clinical data collection: Table 1 shows the clinical and demographic data for the patients. Clinical information on the size, number, stage, and grade of the tumors as well as information about intravesical treatment, dates of recurrence, chemotherapy, radical cystectomy, and pathological outcomes at cystectomy was provided by the urologist in our department. At registration, a blood sample was obtained from each participant in EDTA for genotyping, and it was stored at -80°C.
2.4. Genotyping: Genomic DNA was extracted from peripheral blood cells using a spin column [16]. PSCA C/T gene variant genotyping was performed using predesigned TaqMan assays and the CFX96 Real-Time System Thermal Cycler (Bio-Rad). Positive and negative controls were used for each genotyping test, and 10% of the samples were chosen at random for triple testing with 100% concordance.
2.5. Statistical analysis: To determine the power of the study, a binary logistic regression model was used to assess the risk, and the odds ratio (OR) was calculated with a 95% confidence interval. In the statistical analysis, which was performed with Statistical Package software, version 22.0 (SPSS), P < 0.05 was considered to indicate statistical significance. The goodness-of-fit chi-square test was used to look for deviations from the Hardy-Weinberg equilibrium in the controls.

Table 1

General characteristics of subjects.
VARIABLES		BLADDER CANCER (107)	CONTROLS (105)
GENDER	MALE	84 (78.51)	80 (76.2)
GENDER	FEMALE	23 (21.49)	25 (23.80)
AGE	< 55	31 (28.9)	32 (30.4)
	55–64	24 (22.5)	32 (30.4)
	> 65	52 (48.6)	41 (39.2)
GRADE	LOW	41 (38.3)	-
GRADE	HIGH	66 (61.7)	-
TUMOUR STAGE	SUPERFICIAL	39 (36.4)	-
TUMOUR STAGE	INVASIVE	68 (63.6)	-
OCCUPATIONAL EXPOSURE	YES	57 (53.2)	30 (28.6)
OCCUPATIONAL EXPOSURE	NO	50 (46.7)	75 (71.4)
HISTORY OF UROLOGICAL INFECTION	YES	40 (37.4)	16 (15.3)
HISTORY OF UROLOGICAL INFECTION	NO	67 (62.6)	89 (84.7)

The demographic and tumor characteristics of the subjects are summarized in Table 1. This study population included 107 patients and 105 controls. The proportion of bladder cancer patients with different ages and genders was similar to that of the controls. According to the grade distribution, 41 patients had low-grade tumours (38.3%) and 66 patients had high-grade tumors (61.7%). For occupational exposure, 53.2% of patients in the bladder cancer group were exposed to chemicals and hazardous substances while 28.6% of the subjects in the control group were exposed to chemical and hazardous substances. Approximately 37.4% ofbladder cancer patients had urinary infections in the lower urinary tract.. Table 2 shows the genotype frequencies of rs2294008 among bladder cancer patients and controls and their associations with the risk of bladder cancer. The frequencies of the CC, CT, and TT genotypes of the rs2294008 polymorphism were 26.2%, 42.9% and 30.9% respectively in bladder cancer patients and 51.5%, 30.4% and 18.1% respectively in controls. The genotype frequencies in the controls were in Hardy-Weinberg equilibrium. Compared with the CC genotype, the CT and TT genotypes were associated with an increased risk of bladder cancer (adjusted odds ratio [OR] CT = 2.77, 95% confidence interval [CI] = 1.459–3.247; OR TT = 3.349, 95% CI = 1.610–6.922). Moreover, combined CT/ TT was associated with a significantly increased risk of bladder cancer (OR CT/TT = 2.949, 95% confidence interval [CI] = (1.656–5.252) (Table 2). Table 3 shows the associations between rs2294008 and bladder cancer, which were further evaluated by stratified analysis of tumor grade, and tumor stage. The incidence of rs2294008 CT/TT was greater in high-grade tumors (OR CT/TT = 1.984, 95% CI = 0.892–4.413) than in low-grade tumors (OR CT/TT = 0.452, 95% CI = 0.213–0.961). Similarly, for stage-wise distribution, superficial BC was less effective (T0-T1: OR CT/TT = 0.3367, 95% CI = 0.157–0.721) than invasive BC (T2-T4: OR CT/TT = 2.658, 95% CI = 1.131–6.246). As shown in Fig. 1, the effect of the rs2294008 CT/TT was relatively greater for tobacco exposure (OR CT/TT = 2.374, 95% CI = 1.075–5.243) than for nonsmoking (OR CT/TT = 0.236, 95% CI = 0.101–0.551) with a significant association (p = 0.0001*, p < 0.001). Smoking exposure (OR CT/TT = 2.008, 95% CI = 0.948–4.251) was significantly more strongly associated with smoking than nonsmoking(OR CT/TT = 0.537, 95% CI = 0.243–1.101) with a significant association (p = 0.0001*, p < 0.001)and alcohol exposure (OR CT/TT = 1.643, 95% CI = 0.752–3.589) was not significantly associated with alcohol exposure (OR CT/TT = 0.583, 95% CI = 0.278–1.224) with no significant association (p = 0.0199*, p < 0.001).

Table 2

Genotype Frequencies of PSCA rs2294008 among Bladder cancer and Controls.
Genotype	Controls (105)	Bladder Cancer (107)	Odds Ratio
CC	67(63.8)	53(49.5)	Reference
CT	34(32.4)	46(42.9)	1.71(0.96–3.028)
TT	4(3.8)	8(7.5)	2.52(0.72–8.85)
CT + TT	38(36.2)	54(50.50	1.79(1.03–3.11)
C ALLELE	151(84.7)	129(78.7)	Reference
T ALLELE	25(14.1)	39(23.8)	1.82(1.04–3.17)

Table 3

Subgroup analysis of the Association between PSCA 2294008 and risk of bladder cancer.
TUMOUR GRADE	Odds Ratio		p-value
TUMOUR GRADE	CC	CT/TT	p-value
LOW	1 (Reference)	0.452 (0.213–0.961)	0.001
HIGH	1 (Reference)	1.984 (0.892–4.413)	0.001
TUMOUR STAGE	Odds Ratio
SUPERFICIAL (T0-T1)	1 (Reference)	0.3367 (0.157–0.721)	0.0001
INVASIVE (T2-T4)	1 (Reference)	2.658 (1.131–6.246)	0.0001

Bladder cancer is a worldwide health concern, and it is crucial to identify the genetic elements that impact its onset and progression. The rs2294008 polymorphism in the PSCA gene is a risk factor for BC. It is also the most studied variant in the 8q24.3 region that is linked to susceptibility to BC. This missense variant found in PSCA exon 1 alters a nucleotide in the start codon. The association between rs2294008 and bladder cancer susceptibility was first reported by Wu et al. [17] and subsequently validated by Rothman et al. [18] The per-allele association of rs2294008 was detected in the combined dataset which included10,196 cases and 44,705 controls. OR = 1.13 (95% CI = 1.09–1.17, P = 4.4× 10^− 11). Remarkably, another GWAS on bladder cancer revealed a correlation between the rs2294008 T allele and Caucasian susceptibility to bladder cancer, corroborating this SNP. [17, 18] Although no statistical significance was found, rs2294008 showed a comparable correlation with known risk alleles in Caucasians in a study conducted on a Chinese population [19]. Another Chinese study revealed a correlation between the rs2294008 CT genotype but not the TT genotype and an increased risk of bladder cancer[20]. The dominant genetic model for the combined rs2294008 CT/TT genotypes revealed a statistically significant increase in the risk of bladder cancer. The impact of combined genotype was similar to that observed in Caucasians. (OR CT/TT = 1.38, 95% CI = 1.09–1.75 in Chinese individuals; OR CT/TT = 1.33, 95%CI = 1.22–1.45 in Caucasians) [17, 20]. Additionally, they reported elevated PSCA expression in bladder tumor tissue compared to adjacent normal bladder tissue. (odds ratio [OR] TT = 2.52, 95% CI = 0.72–8.85; OR, CT = 1.71, 95% confidence interval [CI] = 0.96–3.02).

As smoking alters gene expression and damages DNA due to several tobacco carcinogens, smoking is a significant risk factor for bladder cancer [21, 22]. An approximately threefold increased risk of urinary tract cancer was observed among current smokers compared to nonsmokers, as per a meta-analysis that included epidemiological research on urinary tract cancer risk associated with cigarette smoking [23]. The results of a Chinese investigation revealed that smokers with the rs2294008 CC and CT/TT genotypes, had a 2.2 and 3.4 times greater risk of bladder cancer, respectively, than nonsmokers with the CC genotype [20]. Additionally, the combined CT/TT genotype was found to have a greater impact on tobacco exposure among smokers(OR CT/TT = 2.374, 95% CI = 1.075–5.243) than nonsmokers (OR CT/TT = 0.236, 95% CI = 0.101–0.551). We also performed a stratified analysis based on tumor grade and stage, and found that the impact of the combined CT/TT genotype on invasive bladder cancer cancer(OR CT/TT = 0.452, 95% CI = 0.213–0.961) and superficial bladder cancer(T0-T1: OR CT/TT = 0.3367, 95% CI = 0.157–0.721) varied significantly. Similarly, Wang et al. [20] discovered that this genotype was associated with an increased risk of invasive bladder cancer. However, a second study by Lee et al. produced contradictory results [24]. Although the exact cause of the disparity is still unknown, one possibility is that the relatively small sample size of the subgroup analysis prevented it from having enough power. Differential characteristics in the genesis, epidemiology, and pathophysiology of superficial and invasive bladder cancers may provide an additional clarification [25]. To verify these findings, additional studies with larger sample sizes are needed. Overall, our research suggested that rs2294008 in the PSCA gene polymorphism is associated with an increased risk of bladder cancer in the North Karnataka population, suggesting that rs2294008 is involved in the pathogenesis of bladder cancer.

Conflicts of Interest: The authors declare no conflicts of interest.

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No competing interests reported.

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Single Nucleotide Polymorphism in PSCA Associated with Bladder Cancer Susceptibility in an Indian Subpopulation

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Abstract

Background

Materials and methods

Results

Conclusions

Figures

INTRODUCTION

MATERIALS AND METHODS

RESULTS

DISCUSSION

Declarations

References

Additional Declarations

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