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Evaluation of molecular signatures in the urinary bladder and upper tract urothelial carcinomas: a prospective controlled clinical study
Journal of the Egyptian National Cancer Institute volume 34, Article number: 47 (2022)
Urothelial carcinomas (UC) can be either in the upper or in the lower urinary tract or both. Urothelial bladder cancer (UBC) is more common than upper tract urothelial carcinoma (UTUC). This research was designed to study the difference between UBC and UTUC using the molecular pathways including (MAPK/ERK) pathway, cell cycle regulating genes, and oncogenic genes.
To study the discrepancy between UBC and UTUC, a prospective trial was carried out for 31 radical cystectomy and 19 nephrouretrectomy fresh-frozen specimens of UBC and UTUC patients, respectively. The expression level of mRNA of eight genes namely EGFR, ELK1, c-fos, survivin, TP53, RB1, FGFR3, and hTERT was assessed in normal adjacent tissues, UTUC, and UBC by RT-PCR.
Comparison between UTUC and UBC regarding the expression level of mRNA of the EGFR, ELK1, c-fos, survivin, TP53, and FGFR3 had significant difference (p-value < 0.001), while the expression level of RB1 and hTERT level had no significance. Sensitivity/specificity of EGFR, Elk1, c-fos, survivin, TP53, and FGFR3 was 0.78/0.90, 0.84/0.90, 0.84/0.80, 0.84/0.96, 0.94/0.93, and 0.89/0.93, respectively, to differentiate between UTUC and UBC.
Despite the fact that UTUC and UBC share the same origin, there is a clear evidence that there is a molecular difference between them. This molecular difference could be the reason that UTUC is more aggressive than UBC.
Urothelium is the specific epithelial lining of the upper urinary tract that includes the renal pelvis and ureters and lower urinary tract that includes bladder and urethra . Urothelial carcinomas (UC) can be in the upper and/or the lower urinary tract. Urothelial cancers are the most abundant histologic subtype of bladder cancer (BC) with nearly 90% of bladder tumors being urothelial. Upper tract urothelial carcinoma (UTUC) is uncommon, accounting for only 5–10% of all UC .
Both UC of the bladder and upper tract are considered as a disparate twin. Both originates from the same tissue type; however, both have discrepancies in diagnosis, treatment, and prognosis . Both arise from distinct embryonic tissues and have overlapping genomic features .
Previous studies had reported that UTUC was more aggressive than UBC . The tumor aggressiveness is controlled by MAPK\ERK pathway, cell cycle regulators, and oncogenes as they are the major pathways responsible for the tumor invasiveness and metastasis [6,7,8].
MAPK/ERK is a critical pathway for human cancer. It plays an critical role in cell proliferation, differentiation, apoptosis, angiogenesis, and tumor metastasis . Mutations and overexpression of these pathway genes are responsible for the regulation of cell fate, genome integrity, and survival leading to protein amplification, thus overactivating the pathway. These mutations can occur in membrane receptor genes, such as epithelial growth factor receptor (EGFR), and in transcription factors (ELK-1 & c-fos) which had been studied in both tumors separately in previous studies .
Cell cycle pathways involved in carcinogenesis and have a role in tumor prognosis and management. Mutations in cell cycle regulatory genes are the most prevalent in cancer, and they can lead to uncontrolled tumor development and progression. In human urothelial carcinomas, mutations and deregulation of genes involved in the regulation of normal cell cycle progression are common. TP53, retinoblastoma (RB1), and survivin gene mutations are common in UBC and UTUC [4, 7, 10].
Proto-oncogenes have vital functions which are essential for normal homeostasis in cells. Proto-oncogenes are responsible of growth, proliferation, and survival. Tumor cells can then make use of an oncogene’s beneficial properties . Mutated fibroblast growth factor receptor 3 (FGFR3) and hTERT are implicated as the most common oncogenes in the UC .
Several studies had compared between UBC and UTUC in epidemiology, etiology, staging, risk factors, prognosis, and management [1, 2]. Regarding molecular level, UBC is more common and better studied in molecular alterations characterization than UTUC, even in the Cancer Genome Atlas (TCGA) project, likely due to the rarity of the UTUC . There are no enough molecular studies to compare between UBC and UTUC .
Therefore, this study was designed to investigate the molecular pathways in the two types of UC, through shedding the light on EGFR, ELK1, and c-fos from the (MAPK/ERK) pathway; survivin, TP53, and RB1 from the cell cycle regulating genes; and FGFR3 and hTERT from the oncogenic genes.
Patients and methods
This prospective study was performed on pathological specimens of 31 subjects with UBC who underwent cystectomy and 19 with UTUC who underwent radical nephroureterectomy with bladder cuff excision. In each group, fresh-frozen samples were taken from the malignant tissue and non-tumor tissue of the same specimen qRT-PCR. All tumors were graded using the 2004 WHO classification and staged according to the 1997 TNM classification. The institutional review board (IRB) approved this study, and informed consent was taken from all patients.
Real-time quantitative reverse transcription PCR (qRT-PCR)
Total RNA was extracted from frozen tissue specimens using TRIzol (Invitrogen, USA) according to the manufacturer’s protocol. Complementary DNA (cDNA) was synthesized from RNA using high-capacity cDNA reverse transcription kit (Applied Biosystems, USA), following manufacturer’s instructions. qRT-PCR was achieved using SYBER Green PCR Master Mix (Applied Biosystems, USA). The mRNA expression level of EGFR, ELK1, c-fos, survivin, TP53, RB1, FGFR3, hTERT, and GAPDH as the internal control was quantified using step 1 plus real-time PCR (Applied Biosystems). All primers are listed in Table 1. The qRT-PCR program was as follows: initial denaturation for 10 min at 95 °C, then 40 cycles of denaturation for 15s at 95 °C, annealing at 60 °C for 1 min, and finally extension at 72 °C for 1 min. Expression level analysis was done based on the equation (2−ΔΔCT) .
Statistical analysis was performed with the use of the IBM SPSS Statistics ver. 22.0 for Windows (IBM Corp., Armonk, NY, USA). In the normally distributed variables, student t-test was used for comparison between groups. Chi-square and Fisher exact tests were used for comparing categorical data of both groups. Receiver operating characteristic analysis (ROC), an area under the curve (AUC), sensitivity, and specificity were calculated. p-value ≤ 0.05 was considered statistically significant.
Thirty-one subjects with UBC and 19 subjects with UTUC were included in this study. Table 2 shows the patient demographic characteristics of the two groups. Groups were matched in age, sex, BMI, and renal function. Moreover, comorbidities in terms of liver disease, diabetes mellitus, and chronic kidney disease were not significantly different among both groups.
Oncologic comparison between both groups is shown in Table 2. UTUC had significantly lower tumor stage with 89.5% having T1. Moreover, 84.2% of UTUC had organ confined ≤ T2, N0/× versus 38.7% in UBC, a difference of statistical significance. On the hand, lymphovasular invasion was significantly higher in UTUC 73.7% versus 32% in UBC. Meanwhile, both groups were comparable regarding tumor grade, lymph node status, and pathological cell type.
Comparison between the expression level of mRNA of MAPK/ERK pathway including EGFR, ELK-1, and c-fos showed significantly higher levels of expression in both UTUC and UBC compared to normal tissue (P < 0.001). Comparison between UTUC and UBC showed significantly higher levels of expression of the three genes in UTUC (P < 0.001) (Table 3).
Receiver operating characteristic (ROC) curve was obtained for EGFR, ELK-1, and c-fos. The area under the curve (AUC) for EGFR (0.77), ELK-1 (0.94), and c-fos (0.84) was significantly larger than the reference line (P ≤ 0.01). The optimal cutoff values for the investigated biomarkers were determined based on the best balance of sensitivity and specificity. The optimal cutoff values were 3.75, 4.05, and 3.69 for EGFR, ELK-1, and c-fos, respectively (Fig. 1).
Cell cycle regulator
Comparison between the expression level of mRNA of cell cycle regulator survivin and TP53 showed significantly higher levels of expression in both UTUC and UBC compared to normal tissue (P < 0.001). Nevertheless, RB1 expression showed significantly lower level in both UTUC and UBC compared to normal tissue (P < 0.001). UTUC showed significantly higher levels of expression of survivin and TP53 compared to UBC (P < 0.001). Meanwhile, RB1 expression showed comparable results between UTUC and UBC (P = 0.133) (Table 3).
ROC curve was obtained to survivin and TP53. The AUC for survivin (0.84) and TP53 (0.96) was significantly larger than the reference line (P < 0.01). The optimal cutoff values for the investigated biomarkers were determined based on the best balance of sensitivity and specificity. The optimal cutoff values were 7.93 and 4.15 for survivin and TP53, respectively (Fig. 2).
Comparison between the expression level of mRNA of oncogenic genes FGFR3 and hTERT showed significantly higher levels of expression in both UTUC and UBC compared to normal tissue (P < 0.001). UTUC showed significantly higher levels of FGFR3 expression in UTUC compared to UBC (P < 0.001). On the other hand, hTERT showed comparable results between UTUC and UBC (P = 0.395) (Table 3).
ROC curve was obtained for FGFR3. The AUC for FGFR3 (0.89) was significantly larger than the reference line (P < 0.01). The optimal cutoff values for the investigated gene were determined based on the best balance of sensitivity and specificity. The optimal cutoff value for FGFR3 was 3.52 (Fig. 3).
The UTUC and UBC are considered as a disparate twin with distinct putative pathogenetic mechanisms. A molecular study to clarify the discrepancy between them may identify contrasting therapeutic opportunities . The present study investigated (MAPK/ERK) signaling pathway as it plays an important role in the cellular growth differentiation through controlling multiply genes like EGFR, ELK-1, and c-fos.
EGFR has a tyrosine kinase domain which plays an important role in cell signaling pathways involving cell proliferation, apoptosis, angiogenesis, and metastasis through signaling pathways such as MAPK/ERK . Overexpressed or mutant EGFR has a critical role in human cancer including the lung, prostate, and urinary bladder .
In the present study, EGFR was highly expressed in UTUC and UBC tumor specimens compared to normal tissue. This result is consistent with the result reported by Khaled et al.  who found that EGFR is overexpressed in UBC. However, EGFR had significantly higher expression in UTUC than UBC.
ELK1 is a member of transcription factors, regulating the expression of some genes including c-fos and sharing a role in cell apoptosis, migration, and invasion. Immunohistochemical study revealed that levels of ELK1, and its activated form, phospho-ELK1, were overexpressed in bladder tumors . In patients with UTUC, phospho-ELK1 were significantly upregulated, compared with corresponding nonneoplastic urothelial tissues . In the present study, ELK-1 expression was overexpressed in UTUC and UBC tumor tissues compared to normal tissue. On the other hand, ELK1 expression had significantly higher expression in UTUC than UBC.
c-fos is a proto-oncogene that shares a role in the regulation of cell growth, differentiation, proliferation, transformation, and apoptosis. c-fos is significantly expressed in UBC than in adjacent non-cancer tissues . In the present study, c-fos expression had significantly higher expression in UTUC and UBC tumor tissues than the adjacent normal. Huhe et al.  revealed that c-fos was upregulated in UBC using immunohistochemical (IHC). Interestingly, our study showed that the expression level of c-fos was higher in UTUC than UBC.
The present study investigated the cell cycle regulatory genes, as they play a vital role in controlling the replication of apoptosis, and prevents the uncontrolled cell division. The cell cycle is controlled by various genes like survivin, TP53, and RB1.
Survivin is a mitosis regulator and apoptosis inhibitor and promotes angiogenesis. Survivin shows significant overexpression in UBC patients and very low to absent levels in non-cancer . Immunohistochemistry shows that survivin protein overexpression is strongly predictive of recurrence and progression of BC . Altered survivin expression was observed in 39.3% of UTUC tumors and was significantly associated with worse clinicopathological features as advanced stages, lymph node metastases, and lymphovascular invasion . In our trial, survivin was overexpressed in both UTUC and UBC tumor tissues compared to the adjacent normal tissue. It was observed that the level of the survivin expression was significantly higher in UTUC than UBC.
TP53 maintains the genomic stability and activation of cell cycle exit/apoptosis through working as a tumor suppressor. The prevalence of TP53 alterations increases with UBC stage and grade . TP53 was more frequently altered in UBC; however, it was only present in high-grade and high-stage UTUC . TP53 was the second most frequently altered genes in both in UBC and UTUC (58% vs 49%, p = 0.04), respectively . In our study, TP53 is overexpressed in both types of tumor compared to the normal tissue. This result agrees with Khaled et al.  who reported that TP53 was overexpressed in UBC. Also in our study, it was observed that the TP53 was significantly higher in UTUC than UBC.
The RB1 is a key molecular component that regulates cell cycle progression. It controls the G1-S transition in the cell cycle by integrating growth-promoting and suppressing signals. Cancer cells often have mutations in this pathway, which lead to uncontrolled tumor development . In our study, RB1 mRNA expression was low in both UTUC and UBC compared to normal tissue. This result agrees with Khaled et al.  who reported that RB1 is negatively expressed in UBC. We also showed no significant difference between expression of RB1 in UTUC and UBC.
The present study investigated the oncogenic genes that play a role in cell growth and proliferation or inhibition of apoptosis that are controlled by multiple genes like FGFR3 and hTERT.
FGFR3 is a transmembrane tyrosine kinase receptor with auto-phosphorylation activity that controls a variety of physiological processes such as proliferation, differentiation, migration, and apoptosis, and its structural activation is linked to several diseases, including cancer .
Kang et al.  reported that FGFR3 is overexpressed in UBC tissue. In the present study, FGRF3 was found to be overexpressed in UTUC and UBC compared to the adjacent normal tissue. FGFR3 had significantly higher expression in UTUC than UBC.
Telomerase is a ribonucleoprotein enzyme that keeps the telomere length constant by adding telomeric repeats (TTAGGG) to the end of the telomere. The gene hTERT codes for telomerase. In normal human cells, telomerase activity is inhibited, and telomeres are increasingly reduced after each replication cycle, resulting in cell senescence and death . In over 85–90% of malignant cancer cells, telomerase is reactivated and overexpressed, enabling them to survive and proliferate uncontrollably . hTERT was expressed in high level in UTUC and UBC tumor tissue compared to the adjacent normal. Such result was observed in the work of Wu et al.  who reported that hTERT was highly expressed in UTUC tissue. In our study, the comparison between expression of hTERT UTUC and UBC had no significant difference.
The significant expression of EGFR, ELK1, c-fos, survivin, TP53, and FGFR3 in UTUC could explain its aggressive and invasive attitude than UBC . The results of this study showed that EGFR, ELK1, c-fos, survivin, TP53, and FGFR3 are very likely to be involved in the occurrence and development of UTUC. Our study provided references for the molecular etiology of the aggressiveness and invasiveness of UTUC.
The study is limited by having a relatively small sample size and investigating only three molecular pathways of carcinogenesis. Researchers are invited to reproduce this experiment with large sample size and more in-depth studies of large number of molecular pathways. Such larger prospective studies are warranted to consolidate data of our initial results of this preliminary report. Moreover, we advise future researcher to investigate the correlation between the different genes and clinical status of the patient including tumor stage, grade, lymph node status, and metastasis. It might be advisable to use the abnormal expression of the studied genes in gene or target therapy in future studies.
Results of the present study showed clear evidence that there is a molecular difference between UTUC and UBC despite they share the same origin. This molecular difference could be the reason that UTUC is more aggressive than UBC.
Availability of data and materials
All data and material are available if requested.
Area under the curve
Epidermal growth factor receptor
Fibroblast growth factor receptor
Receiver operating characteristic
Real-time polymerase chain reaction
The Cancer Genome Atlas
Urothelial bladder cancer
Upper tract urothelial carcinoma
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We would like to thank the Ministry of Higher Education in Egypt for the financial support of this study.
This work was funded by Ministry of Higher Education, Egypt (Grant no. CEP1-031-MANS).
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The institutional review board (IRB) approval was obtained from the Mansoura Faculty of Medicine (ID number RP.19.07.38), and informed consent was taken from all patients.
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Dawood, E.E., Awadalla, A., Hashem, A. et al. Evaluation of molecular signatures in the urinary bladder and upper tract urothelial carcinomas: a prospective controlled clinical study. J Egypt Natl Canc Inst 34, 47 (2022). https://doi.org/10.1186/s43046-022-00148-x
- Bladder cancer
- Upper tract
- Molecular pathway