Detection of Classic Serum Tumor Markers (CSTMs) Group Benets to Differential Diagnosis and Disease Assessment of UTUC

Background: To explore the potential relationship of changes in classic serum tumor markers (CSTM) with differential diagnosis and disease assessment of upper tract urothelial carcinoma (UTUC). Methods: 60 UTUC (56 operated), 44 RCCC and 36 NTHN, were included into this retrospective analysis. The initial classic serum tumor markers (CSTMs), including CA242, CA199, CA125, CEA, AFP, SCC and CA724, were compared among the three groups. The preoperative, 1 month postoperative and 1 year postoperative/PD parameters (value and abnormal rate) of CSTMs were compared in UTUC group. A recommend test strategy was given and rechecked. The pathological manifestations of tumoral tissues and paracancerous tissues were analyzed. Results: The value of CA242, CA199 and CEA were higher in UTUC than in RCCC. The value of CEA was higher in UTUC than in NTHN. The value of CA199 and CEA were higher in UTUC than in RCCC+NTHN. The AR of CA199, CEA, SCC and CA724 were higher in UTUC than in RCCC. The AR of CEA and CA724 were higher in UTUC than in NTHN. The AR of CA199, CEA, SCC and CA724 were higher in UTUC than in RCCC+NTHN. The preoperative value of CA242 and CA199 were higher than 1 month postoperative ones in UTUC. The 1 year postop/PD value of CA242 and AFP were higher than 1 month postoperative ones in UTUC. For postop PD patients, the value during PD of CA242, CA199 and AFP were higher than 1 month postop ones. The preoperative AR of CA199 and CEA were higher than 1 month postoperative ones in UTUC. The recommended test strategy was given: CA242+CA199+CEA+AFP+SCC+CA724, and was rechecked. The P values were almost the lowest and the positive results covered all the comparisons. In 56 operated cases, the NAC in CSTMs by the recommended test strategy was statistically related with the tumor load. It appeared positive labelling by Ab of CA199, CA125, CEA, AFP and CA724 in UTUC tissues, while negative in paracancerous tissues. Conclusions: CSTMs may help to make differential diagnosis and disease assessment of UTUC. Group test (CA242+CA199+CEA+AFP+SCC+CA724) was recommended and more valuable.


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 nding a diagnostic and prognostic index focused on bladder carcinoma. Some indicators like CxBladder monitor, UroVysion, NMP-22 and bladder tumor antigen were utilized in diagnosis and monitoring of TCC, however, few biomarkers achieve high sensitivity and speci city [1]. And other biomarkers, such as FGFR3, p53, pRb, p21, Ki67 and VEGF, were used as prognostic factors for bladder cancer in previous studies [2][3][4][5][6]. And even some genomic biomarkers were proposed in recent researches [7,8]. However, only a few established prognostic factors were found to effectively assess the tumor progression of upper tract urothelial carcinoma (UTUC) [9]. On the other hand, some classic serum tumor markers (CSTM) 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 [10], seem to have more evaluation functions. In this study, in term of differential diagnosis and disease assessment of UTUC, we analyzed 7 CSTMs commonly utilized in clinical detection, including CA242, CA199, CA125, CEA, AFP, SCC and CA724, which partially presented abnormal in majority of cases. We attempted to explore the potential relationship between CSTMs changes and the UTUC changes.

Ethics statement
All procedures performed in this study involving human participants were in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by institutional ethics committee of Shanghai Ruijin Hospital and informed consent was taken from all the patients.

Study subjects
A total of 140 unilateral affected patients, in which 60 UTUC, 44 RCCC and 36 NTHN (with ipsilateral renal dysfunction), were included into this retrospective, descriptive analysis. In UTUC patients, 56 of them accepted the surgical therapy (radical nephroureterectomy for 53 patients and radical nephrectomy for the rest 3 patients due to the misdiagnose as kidney carcinoma) during the year 2014-2020 in Department of Urology in Ruijin Hospital. The RCCC and NTHN cases were collected as control (in 2018-2019, Ruijin Hospital). All UTUC diagnoses and recurrence, implantation or metastasis diagnoses were con rmed by pathological analysis (biopsy or surgical specimens). There were 1 multiple distant metastasis case and 2 cases of lymphatic metastasis and multiple distant metastasis in preop examinations, to whom the surgical therapy was not suitable. There was also 1 patient without detectable metastasis refused to accept the operation. None of the patients was combined with bladder or urethral transitional cell carcinoma (TCC) in UTUC group. None of the UTUC patients had received radiotherapy, chemotherapy, or immuno-therapy before the initial test of CSTMs. Preoperative metastatic RCCC occurred in 1 patient (iliac joint metastasis) and one case of tumor thrombus in main renal vein.

Methods
The value and the AR of CSTMs were both counted. The comparisons of CSTMs were done among the UTUC, RCCC, NTHN and RCCC + NTHN group, also between preop and 1 month postop, 1 month postop and 1 year postop or progression of disease (PD), 1 month postop and postop PD in UTUC cases. Once a CSTMs had revealed a statistical value in any of the comparisons in this study, it would be gathered into a recommended test strategy. The strategy obtained based on these comparisons would be reanalyzed in all comparisons to compare with the single CSTMs for testing its application value.
The pathological analysis was also collected in 56 UTUC cases which was non-metastatic and have accepted the surgical treatment. The tumor load, grade and in ltration were statistically observed with the value and the NAC of preop CSTMs. The analysis of pathological sections were also speci cally stained for detection of the expression of CA724, CA125, CEA, AFP and CA199, the antibodies (Ab) for immunohistochemical stain of CA242 and SCC were not available.
The tumoral tissues and the paracancerous tissues were pair-compared.

Biochemical detection of classic serum tumor markers
The CSTMs test is as a routine preoperative examen for all patients in our department and also as a routine follow-up examen of the UTUC patients recent years. The serum CA242, CA199, CA125, CEA, AFP, SCC and CA724 levels were detected by electrochemiluminescence immunoassay (Cobas; Roche Diagnostics, Germany) at the Department of clinical laboratory of Ruijin Hospital a liated to Shanghai Jiao Tong University School of Medicine, Shanghai, China. The normal reference values were as follows: CA199 < 35 U/ml, CA242 < 20 IU/ml, CA724 < 8.2 U/ml, CA125 < 35 U/ml, CEA < 5 ng/ml, AFP < 9 ng/ml, SCC < 1.5ng/ml.
In this study, 39 UTUC patients had detected the CSTMs 1 month postoperative and 23 of them had the CSTMs results 1 year postoperative (without recurrence and metastasis) or during PD (with recurrence and metastasis).

Statistical analysis
The status of the level of each CSTM were expressed as mean ± standard error of the mean (SEM). Measurement data between groups were compared with the group t-test, paired t-test, chi-square test (χ2) of a four-fold table, Fischer exact test, Wilcoxon two-sample test, and linear correlation analysis. Statistical analysis was performed using SPSS version 23. All tests were two-tailed and P < 0.05 was considered statistically signi cant.

General parameters
The comparisons of the gender, age, side, BMI, affected renal function and metastasis rate in 3 groups were presented in Table 1.

Strategy of CSTMs test in UTUC
According to the results as above, 6 CSTMs: CA242, CA199, CEA, AFP, SCC and CA724 were recommended to be tested for the UTUC patients. We rechecked the test strategy CA242 + CA199 + CEA + AFP + SCC + CA724 in the comparisons and found that by this strategy, the NAC of CSTMs in UTUC group was signi cantly higher than that in RCCC (P = 0.003), NTHN (P = 0.013) and RCCC + NTHN group (P = 0.001). The preop NAC of CSTMs in UTUC group was signi cantly higher than 1 month postop one (P = 0.0003). The 1 year postop/PD NAC of CSTMs in UTUC group was much higher than 1 month postop one (P = 0.001). The postop PD NAC of CSTMs in UTUC group was signi cantly augmented than 1 month postop in the same patients (P = 0.005). (Table 2, Table 3)

Pathological manifestations and CSTMs in UTUC
In 56 patients with surgical specimens, the NAC in CSTMs by the recommended test strategy was statistically related with the tumor load, which was parameters, like grade, submucosa invasion, muscle invasion, adventitia invasion, renal parenchyma invasion and perirenal fat invasion had no signi cant in uence on the values and the AR of CSTMs, also the NAC in CSTMs by the recommended test strategy, except three statistical differences: (1) the value of CA125 in muscle invaded cases (13.41 ± 10.44 U/mL v.s. 8.56 ± 3.19 U/mL) was much higher than that in muscle non-invaded ones (P = 0.025); (2) the value of AFP in submucosa non-invaded cases (3.77 ± 1.75 ng/mL v.s. 2.66 ± 1.20 ng/mL) was signi cantly higher than that in submucosa invaded ones (P = 0.022); (3) the AR of SCC in adventitia non-invaded cases (25.00 % v.s. 0.00 %) was signi cantly higher than in adventitia invaded ones (P = 0.024). (Table 4) It appeared in UTUC tissues an intense labelling by Ab of CA199 and CEA, a mild labelling by Ab of AFP and CA724, and feeble by Ab of CA125, while the labelling in paracancerous tissues was all invisible (Fig. 1-10).

Discussion
Usually, the UTUC diagnosis is easy, but it occurred sometimes that the UTUC could be misdiagnosed as renal carcinoma, or NTHN. In this study, there were 3 cases (5%) which were diagnosed as renal carcinoma. They accepted the radical nephrectomy instead of classic surgical therapy for UTUC, radical nephrectourectomy. For them, the prognosis was obviously different. And for the UTUC patients, the progression like recurrence and metastasis are not easily detected or de nite diagnosed, even through an imaging examination. So it is needed some simple indicators to differentiate UTUC with other upper urinary tract diseases and to reveal the development of UTUC and curative effect.
Although some indicators like CxBladder monitor, UroVysion, NMP-22 and bladder tumor antigen were used in diagnosis and monitoring of TCC, few biomarkers achieve high sensitivity and speci city [1]. And only a few established prognostic factors were found to effectively assess the tumor progression of UTUC [9]. The expensive and time-consuming nature also restricted new methods' developments. Upon these, there is no clinical recognized bio-indicator for diagnosis, differential diagnosis and status assessment of UTUC.
The classic serum tumor makers (CSTMs, including CA242, CA199, CA125, CEA, AFP, SCC and CA724) are the most commonly used indicators for predicting and monitoring the residual and recurrent of tumors. SCC were used for squamous cell carcinoma predicting [12,13], all other CSTMs were related with digestive system cancer or reproductive cancer [10,[14][15][16][17][18]. However, some unconventional roles were played in CSTMs: CEA and SCC were used for lung cancer diagnosis and prognosis [19,20]; CA125 and CA199 was reported as signi cant markers of endometrium pathology [21]; SCC had a fair diagnostic value for hepatocellular carcinoma [22]. These meant that CSTMs might have more unique values for diagnosis and prognosis of cancerous 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 [23,24].
In our study, we found that the value or AR in the most of the single CSTMs and the NAC in CSTMs group were signi cantly augmented in UTUC than in RCCC, NTHN and RCCC + NTHN, which may help to differentiate the UTUC with other upper urinary tract common diseases, malignant or benign. This also suggested the potential compliant relationship between CSTMs and UTUC. The postop CSTMs parameters were signi cantly lower than the preop ones and have reaugmented in postop PD patients. This revealed that the changes of group of CSTMs complied with the changes of UTUC disease. For the differential diagnosis, CEA and CA724 presented the best ability, but for the disease assessment, CA242 had a best behavior. So it revealed that the single CSTMs could not sacri ce the both functions.
According to the results in all these comparisons, here we have got 6 CSTMs: CA242, CA199, CEA, AFP, SCC and CA724, which were related with UTUC. A test strategy based on these 6 CSTMs was recommended in the clinical detection. The relationship between this strategy and differential diagnosis and assessment of UTUC was re-check in the comparisons above, the P values were almost the lowest and the positive results covered all the comparisons. So the recommended strategy was considered practical, and the AR seemed to be more important than the value.
In pathological level, tumor load, grade, invasive depth, seemed like no signi cant relationship with most of the single CSTMs, the same to grade and invasive depth with CSTMs group. The NAC on CSTMs by the recommended strategy was linear correlated with tumor load. All these meant that the CSTMs were related with tumor cell quantity, the CSTMs were produced by tumor cell since perhaps the beginning, and the NAC on CSTMs might be a good indicator to assess the changes of disease.
The immunohistochemical labelling results con rmed the fact that the UTUC tissues, but not the paracancerous tissues, signi cantly contained the CSTMs, at least CA199, CA125, CEA, AFP and CA724. In which, CA199 and CEA were strongly expressed, while CA125 was expressed with very small amount (but also much more strongly than paracancerous tissues). These results were highly in accord with the previous biochemical results and the recommended strategy based on the biochemical results. According to these all above, the CSTMs were specially secreted by the UTUC cell, and the recommended test strategy was practical. This explained the compliance of the CSTMs changes with UTUC disease changes.