Elevation of serum tumor markers caused by hydronephrosis due to upper tract urothelial carcinoma

The aim of this study was to explore the association of changes in classic serum tumor markers and pathological and clinical manifestations of upper tract urothelial carcinoma (UTUC) and to analyze its possible mechanism. A retrospective, descriptive analysis was performed in consecutive patients who were diagnosed UTUC during 2014 – 2018 year in our hospital. Detection of classic serum tumor markers (CSTMs, including CA199  CA242  CA724  CA125  CEA  AFP and SCC-Ag) and pathological manifestations of tumor was collected. The status of hydronephrosis caused by tumor, tumor load and the expressions of CA199, CA724 and CEA in UTUC was also analyzed. Higher rate of abnormal changes in CSTM was found in UTUC patients and it could be dropped off after surgical treatment. There was a correlation between UTUC and the classic serum tumor markers including CA199  CA242  CA724 and CEA. Tumor metastasis and hydronephrosis induced by tumor may be two independent reasons which caused this phenomenon. Immunohistochemistry technology proved CA199, CA724 and CEA were almost expressed in almost all UTUC tissues. We suggested a hypothesis that the pressure acting on tumor caused by hydronephrosis may extrude tumor markers produced from UTUC into blood and even promote metastasis. This may be helpful for distinguished the than in high grade UTUC, but higher in non-infiltrating UTUC than in infiltrating UTUC. grade and two indicators of poor prognosis, but take contrary conclusions about abnormal CSTMs changes. There may be other special factor which could induce abnormal CSTMs changes. We proposed pressure caused hydronephrosis one reasonable


Abstract
The aim of this study was to explore the association of changes in classic serum tumor markers and pathological and clinical manifestations of upper tract urothelial carcinoma (UTUC) and to analyze its possible mechanism. A retrospective, descriptive analysis was performed in consecutive patients who were diagnosed UTUC during 2014 -2018 year in our hospital. Detection of classic serum tumor markers (CSTMs, including CA199 CA242 CA724 CA125 CEA AFP and SCC-Ag) and pathological manifestations of tumor was collected. The status of hydronephrosis caused by tumor, tumor load and the expressions of CA199, CA724 and CEA in UTUC was also analyzed. Higher rate of abnormal changes in CSTM was found in UTUC patients and it could be dropped off after surgical treatment. There was a correlation between UTUC and the classic serum tumor markers including CA199 CA242 CA724 and CEA. Tumor metastasis and hydronephrosis induced by tumor may be two independent reasons which caused this phenomenon. Immunohistochemistry technology proved CA199, CA724 and CEA were almost expressed in almost all UTUC tissues. We suggested a hypothesis that the pressure acting on tumor caused by hydronephrosis may extrude tumor markers produced from UTUC into blood and even promote metastasis. This may be helpful for distinguished the upper urinary tract tumor without definite diagnosis.

Background
Urinary epithelial transitional cell carcinoma (TCC) includes renal pelvic cancer, ureteral cancer, bladder cancer, and urinary tract cancer. Among them, most studies aimed at finding a prognostic index focused on bladder carcinoma. A variety of biomarkers, such as FGFR3, p53, pRb, p21, Ki67 and VEGF, were used as prognostic factors for bladder cancer in previous studies [1][2][3][4][5]. And even some genomic biomarkers were proposed in recent researches [6,7]. However, only a few established prognostic factors were found to effectively assess the tumor progression of upper tract urothelial carcinoma (UTUC) [8]. On the other hand, some classic serum tumor markers such as carbohydrate associated cancer antigen 199 (CA199), cancer antigen 125 (CA125), carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP), which were widely used for the diagnosis of different types of gastrointestinal cancer [9], seem to have more prognostic functions. In this study, we detected 7 classic serum tumor markers (CSTM, including CA199, CA242, CA724, CA125, CEA, AFP and SCC-Ag) in 40 patients with UTUC, 37 patients with non-tumoral hydronephrosis, and 44 patients with renal cell carcinoma. We attempted to explore the association between classic serum tumor markers and tumor progression of UTUC, and tried to explain this phenomenon.

Measurement data
All patients received triphasic enhanced dynamic CTU scanning. A neoplasm consisting of tumor cells obstructed calyce or ureter, leading to a focal hydronephrosis. The dilation of hydronephrosis was assessed as hydrop length (HL) using the border diameter between extrarenal and intrarenal renal pelvis (for ureteral TCC) or longest diameter of hydrop renal calyce (for renal pelvic TCC) measured in kidney CT scanning ( Figure 1). Tumor load was assessed by tumor volume calculating with the maximum tumor dimension in three orthogonal planes (length, width, and height measuring from kidney CT scanning image or surgical specimen). Calculating formula is × Length × Width × Height [10].

Immunohistochemistry technology
Immunohistochemical staining of CA199, CA724 or CEA was performed to UTUC tissues of 4 patients picked randomly. The specimens were fixed in 10% buffered formalin, embedded in paraffin and cut into 5 μm sections. Automatic immunohistochemical instrument (LeicaBOND III, Australia) was used for immunohistochemical staining according to the instructions. CA 199 (Leica, Art.201902001, England) CA 724 (Abcam, ab199002, USA) and CEA (Leica, Art.201809001, England) were used for staining respectively. Light microscopy was used for observation at a magnification of ×100.

Statistical analysis
The status of hydronephrosis and the level of each CSTM were expressed as mean ± SD.
Measurement data between groups were compared with the t test or Chi-square test (for categorical variables). Statistical analysis was performed using SAS version 8 statistical software and artwork was created with GraphPad Prism version 6. All tests were two-tailed (some tests were showed one-tailed as well) and P < 0.05 was considered statistically significant.

The abnormal changes in CSTMs were severer in UTUC patients and it could be dropped off after surgical treatment
We analyzed all abnormal changes of CSTM in UTUC, simple hydronephrosis with no carcinoma and renal cell carcinoma patients. The positive rate of abnormal changes in CSTM was higher in UTUC patients than that in other two groups (Table 1). We especially take a comparison between ureteral TCC and simple hydronephrosis with no carcinoma patients as they shared same characteristic on hydronephrosis. We found that although hydrops of renal pelvis in simple hydronephrosis patient was more serious than that in ureteral TCC patient, the positive rate of abnormal CSTM changes in ureteral TCC showed significantly higher than that in simple hydronephrosis (Table 2). These results indicated that UTUC was a leading cause of abnormal CSTM changes but not the hydronephrosis or other renal tumor.
Totally 19 UTUC patients had underwent surgical treatment in our hospital. CSTMs changes in these patients before and after surgery were compared (Table 3). We found the number of abnormal CSTM changes was decreased after surgery (P = 0.02). The rate of abnormal CSTM changes was decreased but with no statistical significance, which may due to that just one indicator was abnormal would seemed as positive even if actual value had dropped.

UTUC pathological progression may effect abnormal CSTM changes
Tumor metastasis were confirmed by CT scanning, including invasion of adjacent tissues and lymph node metastasis. UTUC patients were divided into four group according to metastasis status and tumor site: non-metastasis ureteral TCC, non-metastasis renal pelvic TCC, metastasis ureteral TCC, metastasis renal pelvic TCC. As presented in the Table 4, rate of abnormal changes in the CSTMs was found in metastatic UTUC patients (6 in 6, 100%) and non-metastatic UTUC patients (18 in 34, 51.43%) with statistically difference between two groups (one side P = 0.04). These results suggested that UTUC metastasis may exist correlation with abnormal CSTM changes.
As all metastasis UTUC patients presented abnormal CSTM changes, we analyzed the effect of tumor grade and tumor infiltration on CSTM changes in non-metastasis UTUC patients. According to the result of tissue biopsy, patients were divided into four groups: low grade UTUC, high grade UTUC, noninfiltrative UTUC, infiltrative UTUC. As presented in the Table 5, for non-metastatic UTUC patients, the rate of abnormal changes in CSTMs in low grade UTUC was significantly increased than that in high grade UTUC (P = 0.02), as well as higher in non-infiltrative UTUC than that in infiltrative UTUC (P = 0.02). These results suggested that low tumor grade may promote abnormal CSTM changes, and noninfiltrative tumor may had stronger effect on rising CSTM levels.
The association between tumor load and CSTM levels (

Hydronephrosis may be one factor on abnormal CSTM changes
We tried to find the association between hydronephrosis status and abnormal CSTM changes in nonmetastasis UTUC patients (Table 7)

Serum level of CA199, CA242, CA724 and CEA may be helpful for UTUC prognosis
We showed all 7 CSTM testing results and status of hydronephrosis in Table 9. There is no abnormal result of CA125 or AFP in UTUC patients, so no comparison was did in this

CA199, CA724 and CEA were expressed in almost all UTUC tissues
The expressions of CA199, CA724 and CEA in UTUC tissues were as anti-CA242 antibody was failed to obtained. 4 different UTUC tissues of patients were picked randomly from participates included in this study. As showed in figure 2, Almost all UTUC tissues were expressing CA199, CA724 and CEA more or less. This may verified the abnormal levels of CSTMs may be originated from UTUC tissues.

Discussion
Classic serum tumor makers (CSTMs, including CA199 CA242 CA724 CA125 CEA AFP and SCC-Ag) had been one of the most important indicators for predicting and monitoring the residual and recurrent of tumor. Actually, expect SCC-Ag were used for squamous cell carcinoma predicting [11,12], all other CSTMs were related with digestive system cancer or reproductive cancer [13][14][15][16][17].
However, some unconventional roles were played in CSTMs: CEA and SCC-Ag were used for lung cancer diagnosis and prognosis [18,19]; CA125 and CA199 was reported as significant markers of endometrium pathology [20]; SCC-Ag had a fair diagnostic value for hepatocellular carcinoma [21].
These meant that CSTMs may have more unique values for diagnosis and prognosis of diseases.
However, among these 7 CSTMs, only CEA had been reported as early sign of malignancy in urotheliomas of the upper urinary tract and closely related to the recurrence and survival [22,23].
Some indicators like CxBladder monitor, UroVysion, NMP-22 and bladder tumor antigen were used in diagnosis and monitoring of UC, however, few biomarkers achieve high sensitivity and specificity [24].
The expensive and time-consuming nature was also restricted new methods' developments. On the other hand, UTUC showed several different characteristics compared to bladder cancer at molecular mechanisms underlying tumor development and progression or tumor behavior [25,26]. Upon these, there is no clinical recognized indicator for diagnosis and prognosis of UTUC.
In our study, we found some of CSTMs existed correlation with UTUC, which may help urologists to assessed mass-like lesions in renal pelvis and ureter . Although indicators of change are not the same in renal pelvic TCC or ureteral TCC, the number of changes in CSTMs showed well-predicting use.
Tumor load seemed like no relationship with abnormal CSTMs. Abnormal CSTMs changes existed in all metastatic UTUC patients. Interestingly, in non-metastatic UTUC patients, the rate of abnormal changes in CSTMs in low grade UTUC was significantly increased than that in high grade UTUC, but higher in non-infiltrating UTUC than that in infiltrating UTUC. Low grade carcinoma and tumor infiltration were two indicators of poor prognosis, but take contrary conclusions about abnormal CSTMs changes. There may be other special factor which could induce abnormal CSTMs changes. We proposed pressure caused by hydronephrosis could be one reasonable factor.
We found a positive correlation existed between the severity of hydronephrosis and the number of biomarkers produced by carcinoma cells was not easy to get into blood. As hydronephrosis induced by UTUC became more and more serious, the pressure not only dilated renal pelvis and ureter but also caused compression on tumor, which may induce nonspecific elevation of CSTMs. This could also explain why abnormal CSTMs changes were less in non-infiltrating UTUC patients than that in infiltrating UTUC patients: non-infiltrating carcinoma was often exophytic growth which could induced more serious hydronephrosis. Furthermore, also there was no direct evidence showed that high pressure in renal pelvis and ureter caused by transitional cell carcinoma in upper urothelial tract may increase the risk of tumor metastasis, urologists should be vigilant on this possibility.
There are some limitations in our study. First, the sample size in this research was small as our hospital has just been established for 5 years and cases accumulation was limited. Further accumulation of cases and accurate researches should be carried out to confirm results upon. Second, as not all UTUC patients accepted surgery therapy and limited cases size, analysis of CSTMs changes before and after surgical treatments was oversimplify, and only a few tissues of patients were accepted immunohistochemistry stain. We used CT scanning result to assessed the severity of hydronephrosis but it could not be reflect the pressure in renal pelvis and ureter in accuracy. The results must be validated in large, independent cohorts before it can be applied generally.

Conclusion
There may be still a correlation between transitional cell carcinoma in UUT and the classic serum tumor markers like CA199 CA242 CA724 and CEA. Tumor metastasis may effect this phenomenon, and high pressure induced by hydronephrosis may be one reason of abnormal CSTMs changes.
Hydronephrosis caused by UTUC was associated with poor prognosis and whether it may induced tumor metastasis need further study.

Consent for Publication
Not applicable.

Availability of data and material
The datasets during and/or analyzed during the current study available from the corresponding author on reasonable request.

Competing interests
No competing interests in this research.      Chi-square test was used for comparison for each two groups. The liner regression analysis was used for comparison for each two groups.  T test was used for comparison for each two groups. The liner regression analysis was used for analyzing the hydronephrosis status and the number of abnormal CSTM changes in each group, P values were presented upon. Figure 1 The dilation of hydronephrosis was assessed as hydrop length using the border diameter between extrarenal and intrarenal renal pelvis (for ureteral TCC, Left) or longest diameter of hydrop renal calyce (for renal pelvic TCC, Right) measured in kidney CT scanning.

Figure 2
The expressions of CA199, CA724 and CEA in UTUC (at a magnification of ×100).
Immunohistochemistry technology was used to verified CA199, CA724 and CEA were common expressed in UTUC tissue.