INTRODUCTION
Worldwide, bladder cancer is the 6th most common cancer in men and the 17th most common cancer in women. Notably, in the developed world, bladder cancer ranks the 4th and 9th most common cancer in men and women, respectively [1]. Urothelial carcinoma is the most frequently occurring type of bladder cancer.
Bladder cancer-related mortality is associated with the development of metastasis. Bladder cancer arises because of multistep alterations, among which metastasis is crucial. The treatment of bladder cancer is based on multiple parameters, including the extent of the disease, the bladder cancer stage, and the results of a bladder cystoscopy, which evaluates the tumor. The currently available treatments are not effective, and the mechanisms of metastasis and progression of bladder cancer remain unresolved [2].
The chemokine receptor CXCR4 belongs to the large superfamily of G protein-coupled receptors and has been identified to play a crucial role in several biological processes, including the trafficking and homeostasis of immune cells such as T lymphocytes. CXCR4 has also been found to be a prognostic marker in various types of cancer, including leukemia and breast cancer, and recent evidence has highlighted the role of CXCR4 in prostate cancer. Furthermore, CXCR4 expression is upregulated in cancer metastasis, leading to enhanced signaling. These observations suggest that CXCR4 is important for the progression of cancer [3,4].
The matrix metalloproteinases (MMPs), a family of zinc-dependent endopeptidases with proteolytic activity against extracellular matrix components, are involved in numerous physiological processes, including tissue remodeling, embryonic development, and reproduction.
The overexpression of MMPs is observed in several
CXCR4 and MMP-9 Immunoexpression are Associated with Metastasis in Infiltrating Urothelial Carcinoma of The Bladder
Siska Dwiyantie Wahyuni
*, Sri Suryanti, Hasrayati Agustina, Bethy Suryawathy Hernowo
Department of Anatomical Pathology, Faculty of Medicine, Universitas Padjadjaran/ Hasan Sadikin General Hospital, Bandung, Indonesia
A R T I C L E I N F O
Received : 25 November 2021 Reviewed : 24 December 2021 Accepted : 11 March 2022 Keywords:
CXCR4, infiltrating urothelial carcinoma of the bladder, metastasis, MMP-9
A B S T R A C T
Background: Infiltrating urothelial carcinoma of the bladder (IUCB) is an aggressive urinary tract cancer. Most of the patients were diagnosed with advanced stages, and many have metastasized.
Metastasis is responsible for 90% of cancer deaths compared to the primary tumor itself. In previous research, the expression of CXCR4 and MMP-9 has been suggested to correlate with metastasis and prognosis in several cancers. This study aimed to analyze the association of CXCR4 and MMP-9 expression with metastasis in infiltrating urothelial carcinoma of the bladder.
Methods: This cross-sectional study included 40 selected paraffin-embedded tissue blocks from histopathologically diagnosed IUCB patients, consisting of 20 cases of non-metastasizing and 20 cases of metastasizing IUCB. Immunohistochemical staining for CXCR4 and MMP-9 was performed in all cases. All data were analyzed using the Chi-Square test with p < 0.05 of a significant level and then processed using SPSS 24.0 for Windows.
Results: High CXCR4 immunoexpression was found in 18 (45%) of all cases, and high MMP-9 immunoexpression was found in 19 (47.5%) cases. High immunoexpression of CXCR4 and MMP- 9 in infiltrating urothelial carcinoma of the bladder showed significant association with metastasis (p < 0.05). The stepwise logistic regression analysis revealed that both CXCR-4 and MMP-9 immunoexpressions were independent prognostic factors for metastasis.
Conclusions: CXCR4 and MMP-9 play a role in the metastatic process in infiltrating urothelial carcinoma of the bladder.
*Corresponding author:
Siska Dwiyantie Wahyuni
Department of Anatomical Pathology, Faculty of Medicine, Universitas Padjadjaran/ Hasan Sadikin General Hospital, Bandung, Indonesia [email protected]
Statistical analysis
The association of CXCR4 and MMP-9 with metastasis in IUCB was statistically evaluated using the Chi-square test while the logistic regression test was used for multivariate analysis. The data were considered significant if p < 0.05. Data processing in this study was performed using Statistical Package for Social Science (SPSS) version 24.0 for Windows.
RESULT
Forty patients were included in this study with a mean age of 57.76 years. The age range was 36–82 years. Most of the patients were male (90%) as shown in Table 1.
Most of them were at T3–T4 (82.5%). The number of T4 stage was nearly the same as the number of T3 (42.5%
vs 40%) [10].
Table 2 describes the comparison of characteristics by age, sex, and T stage in the metastatic and non- metastatic groups. There was no difference in the mean age and sex of both the metastatic and non-metastatic groups. The statistical test results obtained a p-value of 0.790 and 1.000, consecutively, which means no association between age and sex with metastasis in IUCB.
There was a difference in the proportion of T stage in the metastatic and non-metastatic groups. The results of statistical tests obtained a p-value of 0.035 (< 0.05) which means, in this study, T stage is associated with metastasis on IUCB.
Based on Table 3, high CXCR4 immunoexpression was higher in the metastasis group compared to the non-metastasis (p = 0.001) This showed that there was a statistically significant association between CXCR4 immunoexpression and metastasis in IUCB.
Table 1. Clinicopathological characteristic of infiltrating urothelial carcinoma of the bladder
Variable N (%)
Age (year)
Mean ± SD 57.76 ± 11.088
Sex
Male 36 (90%)
Female 4 (10%)
T Stage
T1 0 (0%)
T2 7 (17.5%)
T3 16 (40%)
T4 17 (42.5%)
diseases. Among the MMP class, MMP-9 was most frequently involved in tumor aggressiveness, such as invasion and metastasis. Currently, MMP-9 has been shown to overexpress several malignancies such as breast, prostate, lung, colorectal, gastric, thyroid, and other cancers [2,5-7]. This study aimed to analyze the association of CXCR4 and MMP-9 expression with metastasis in infiltrating urothelial carcinoma of the bladder.
METHODS
This study employed an analytical observational research design using the cross-sectional method.
Sampling was conducted using the consecutive sampling technique and taken from the hospital blocks of patients at Dr. Hasan Sadikin Hospital, Bandung, Indonesia, from January 2014 to April 2020. Based on Hosmer and Lemeshow sample formula from sample size 2.0, there were 40 selected paraffin blocks consisting of 20 cases of non-metastasizing and 20 cases of metastasizing IUCB, which met the inclusion and exclusion criteria. Inclusion criteria included the paraffin blocks undergoing biopsies and surgery and diagnosed with histopathologically diagnosed IUCB, with no missing data. Exclusion criteria included paraffin blocks with an insufficient tumor mass.
Metastasis was defined by the presence of tumor masses based on histopathological and radiological findings.
Immunohistochemical examination
Immunohistochemical staining for CXCR4 and MMP-9 was performed in all cases. Each paraffin block was cut to 4 microns thick and deparaffinated with xylol and alcohol. Antigen retrieval was performed using decloaking chamber at 95 °C for 30 minutes. Immunohistochemical staining in this study was done with labeled streptavidin- biotin immunoperoxidase complex. The primary antibodies used were rabbit polyclonal antibody anti- CXCR4 GTX13854 (Genetex) in 1:150 dilution and rabbit polyclonal antibody anti-MMP-9 GTX100458 (Genetex) in 1:150 dilution. The secondary antibody used was Starr Trek Universal HRP Detection STUHRP700L10-KIT (Biocare).
The assessment of CXCR4 and MMP-9 immunoexpression was carried out using the histoscore method. The assessment was done by observing its intensity and staining distribution. The result was regarded as positive if the cell membrane or cytoplasm of tumor cells turned brown.
Staining intensity was rated 0 (negative), +1 (weak), +2 (moderate), or +3 (strong). The percentage of positive staining tumor cells (distribution score) was assigned as 0 (0%), 1 (< 10%), 2 (10–50%), 3 (51–80%) and 4 (> 80%).
The histoscore was obtained by multiplying the intensity and distribution scores. Histoscores of 0–7 were assigned as low immunoexpression and 8–12 as high immunoexpression. The results of CXCR4 and MMP-9 were blindly assessed by two pathologists using Olympus CX21 light microscope [8,9].
Variable
Metastatic Status
P value Metastatic Non Metastatic
N (%) N (%)
Age (year)
Mean ± SD 57.30 ± 8.392 58.25 ± 13.471 0,790
Sex 1,000
Male 18 (90%) 18 (90.0%)
Female 2 (10%) 2 (10.0%)
T stage 0,035*
T1 0 (0%) 0 (0%)
T2 1 (5%) 6 (30%)
T3 6 (30%) 10 (50%)
T4 13 (65%) 4 (20%)
Numerical data for age were tested using an unpaired T-test. Data categorical gender and T stage were tested using Fisher’s exact test, * P value <0.05 means significant or statistically significant.
Table 2. Comparison of the characteristics of research subjects based on the metastatic and non-metastatic status groups
Variable
Metastatic Status
Total OR CI (95%) P value N (%)No Yes
N (%)
CXCR4 Immunoexpression 0.001*
High 4 (20%) 14 (70%) 18 (45%) 9.333
Low 16 (80%) 6 (30%) 22 (55%) (2.180 - 39.962)
P value is calculated with Chi-squared test. * sign indicates p-value of p<0.05. Meaning statistically significant.
Table 3. Association of CXCR4 with metastatic on infiltrating urothelial carcinoma of the bladder
Variable
Metastasis Status
Total OR CI (95%) P value N (%)No Yes
N (%)
MMP-9 Immunoexpression 0.004*
High 5 (25%) 14 (70%) 19 (47.5%) 7.000
Low 15 (75%) 6 (30%) 21 (52.5%) (1.739 - 28.174)
P value is calculated with Chi-squared test. *P value<0.05 means significant or statistically significant.
OR, odds ratio; CI, confidence interval Table 4. Association
of MMP-9 with
metastatic on infiltrating urothelial carcinoma of the bladder
Variable P value OR CI 95%
Lower Upper
Multivariate
T Stage 0.101 0.327 0.086 1.241
CXCR4 0.054 6.526 0.966 44.090
MMP-9 0.014 10.059 1.603 63.129
Multivariate analysis with binary logistic regression. The independent variable included in the logistic regression model is all independent variables with P value less than 0.25 in bivariate analysis.
OR, odds ratio; CI, confidence interval Table 5. Multivariate analysis
of T Stage, CXCR4, and MMP-9 immunoexpression
Figure 1. CXCR4 Immunoexpression. (A) Strong intensity;
(B) Moderate intensity; (C) Weak intensity. MMP-9 Immunoexpression; (D) Strong intensity; (E) Moderate intensity; (F) Weak intensity
Based on Table 4, high MMP-9 immunoexpression was higher in the metastasis group compared to the non-metastasis (p = 0.004) This showed that there was a statistically significant association between MMP-9 immunoexpression and metastasis in IUCB.
Table 5 showed that CXCR4 and MMP-9 immunoexpression is independently associated with metastasis in IUCB, where T stage becomes a dependent prognostic marker. In a further step of multivariate analysis, CXCR4 and MMP-9 expression retained their negative impact on the metastasis of IUBC. MMP-9 showed a stronger association with metastasis compared to CXCR4.
CXCR4 and MMP-9 Immunohistochemical staining were performed in all samples. The staining result was evaluated based on the intensity and distribution of the stained tumor cells (Figure 1).
DISCUSSION
Infiltrative urothelial carcinoma of the bladder (IUCB)- related mortality is associated with the development of the metastatic potential of primary tumor lesions [1]. The chemokine receptor CXCR4 and MMP-9 is a prognostic marker in various types of cancer, including breast cancer, prostate, and thyroid. IUCB preferentially metastasizes to the lung, bones, liver, and lymph nodes, all of which represent organs that secrete high levels of CXCL12. CXCL12 acts as a chemoattractant that drives CXCR4-positive primary tumor cells toward secondary metastatic sites leading to the onset of metastatic lesions [11,12]. Matrix metalloproteinases 9, which represent the most prominent family of proteinases associated with tumorigenesis, are a family of zinc- dependent endopeptidases. The degradation of the basement membrane and extracellular matrix (ECM) by MMPs facilitates tumor cell invasion and proliferation in the metastatic environment. The basement membrane and ECM, on one hand, provide substrates and nutrition for tumor growth and metastasis, but they are major blockades in the prevention of tumor cell invasion and metastasis [7]. During the degradation of the ECM and
basement membrane, MMP 9 is the most important enzyme and plays a key role in this degradation process.
The expression and activation of MMPs are also involved in various physiological and pathological events, such as inflammation, tissue fibrosis, angiogenesis, invasion, and tumor metastasis [2,5-7].
CXCR4 is a pair of G-protein receptors with seven transmembrane helices. CXCR4 expression has been shown to play an important role in migration, metastasis, and poor prognosis in malignancy. Although the exact mechanism of CXCR4 activity has not been fully explained, CXCR4 can induce or support carcinogenesis through the interaction of CXCR4 with CXCL12 ligand, which mediates the activation of phosphatidylinositol 3-kinase (PI3K) and AKT, resulting in cell proliferation and survival. The activated CXCR4-CXCL12 axis also activates signals from MAPK that promote chemotaxis and proliferation. CXCR4- CXCL12 axis also activates phospholipase C (PLC)/protein kinase C (PKC)-Ca2+ signals that promote cell migration and metastasis [11,12].
In this study, CXCR4 immunohistochemical staining was performed in both groups of metastatic and non- metastatic infiltrating urothelial carcinoma. The result showed CXCR4 immunoexpression and the occurrence of metastasis in IUCB (p = 0.001) with OR = 9.333. This result indicates that IUCB patients with high CXCR4 immunoexpression are nine times as likely to be associated with metastasis as IUCB patients with low CXCR4 immunoexpression. The higher CXCR4 immunoexpression means the higher tumor cell’s ability to undergo metastasis. The result of this study is consistent with the study conducted by Wang et al.
[13] showing that overexpression of CXCR4 of bladder carcinoma plays a significant role in the process of invasion and metastasis (p < 0.05). A study conducted by Retz et al. [14] stated that CXCR4 overexpression in bladder carcinoma correlates with the potential for migration and metastasis (p = 0.018).
Matrix metalloproteinase 9 (MMP 9) is an MMP family, that weighs 92 kDa, and is the main enzyme for degrading type IV collagen found on the basement membrane, supporting invasion and metastasis [15,16].
The result of this study showed that MMP-9 immunoexpression is associated with metastasis of IUCB.
The result showed MMP-9 immunoexpression and the occurrence of metastasis in IUCB (p = 0.004) with OR
= 7.000. This result indicates that IUCB patients with high MMP-9 immunoexpression are seven times as likely to be associated with metastasis as IUCB patients with low MMP-9 immunoexpression. The higher MMP-9 expression means a higher probability of metastasis occurs. This result is consistent with the result of a study conducted by Wu et al. [17] on breast carcinoma stating high MMP-9 immunoexpression correlates with the potential for lymph node metastasis. A study conducted by Cai et al. [7] showed that MMP-9
Competing of Interest
The authors declare no competing interest in this study.
Acknowledgment Not applicable.
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overexpression in lung adenocarcinoma was linked to invasion and metastasis.
In this study, multivariate analysis showed both CXCR4 and MMP-9 have p < 0.05, which means that both are significant factors for metastatic status and are independent factors so that they can be called independent prognostic factors for metastasis in IUCB.
It means that both markers can predict metastasis in IUCB. Each marker can become a predictive marker, so we can use just one marker to predict metastasis in IUCB.
This study has several limitations, including a small number of samples, the assessment of expression using only immunohistochemistry, a lack of comparison between metastatic sites, and the use of only single random sampling. The disparity of confidence interval because of the small sample and the method of estimating population values (parameters) using single random sampling was conducted because direct calculations on the entire population are not possible.
Meanwhile, the confidence interval is the value of the range through the actual value that occurs in the population. The results obtained from the sample are still estimates, meaning that they are not true in absolute terms, so it is still possible to vary between samples, which can be shown through the value of this confidence interval. The disparity was found to be caused by tumor heterogeneity, differences in sampling locations of tumors, examining either primary or metastatic locations, and ethnic differences.
The authors recommend more comprehensive studies with a larger sample size taking a cohort analysis approach by using PCR gene expression measurement, taking tumor samples from multiple sites or multiple site sampling, and comparing primary and metastatic sites to address tumor heterogeneity.
CONCLUSIONS
There was a significant correlation between CXCR4 and MMP-9 expression with metastasis on infiltrating urothelial carcinoma of the bladder. Increased CXCR4 and MMP-9 expression were associated with a higher possibility of metastasis in infiltrating urothelial carcinoma of the bladder. These findings suggest that the high expression of CXCR4 (histoscore ≥ 8) and MMP- 9 (histoscore ≥ 8) can be considered to predict metastasis on infiltrating urothelial carcinoma of the bladder.
DECLARATIONS
Ethics ApprovalThis study was approved by the Health Research Ethics Committee of the Faculty of Medicine Universitas Padjadjaran (LB.02.01/X.6.5/222/2020).
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