Glycosaminoglycans in the Urinary Bladder Mucosa, Tumor Tissue and Mucosal Tissue around Tumor

Introduction/Aim. Glycosaminoglycans (GAG) are one of the main constituents of the connective tissue and cellular membrane. Their presence has been evidenced in mucosa and muscular tissue of the urinary bladder of both healthy individuals and those affected by carcinoma. This suggest their potential role in the onset of bladder carcinoma and follow-up of those patients. The aim of the study was to determine GAG levels in tumor tissue and the surrounding bladder mucosa in patients with bladder tumor, as well as in the bladder mucosa in patients with bladder carcinoma, and to compare the results according to the grade and stage of tumor and relapse. Methods. Tissue samples were taken in 61 patients (48 males and 13 females), mean age 61.5 years, range 40–92 years, obtained by transurethral resection (TUR) of bladder tumor, and 8 healthy persons. Determination of a total GAG content in the tissue samples was done by the Whiteman ' s method and then compared regarding the tumor grade and stage. Results. Tumor grade and stage directly correlated with the levels of GAG. The GAG levels were significantly higher in tumor samples as compared to healthy mucosa. Conclusion. Higher GAG levels were recorded in all the patients with bladder tumors comparing to smples obtained from healthy individuals. GAG levels do not predict tumor relapse.


Introduction
Urinary bladder carcinoma is quite often asymptomatic and at the moment of detection invasive carcinoma is already present in 25% of cases.Out of the remaining 75% of superficial forms in about 15% of cases progression into invasive carcinoma occurs.
The risk of tumor progression and relapse is paralelly increased with higher clinical stage and grade as well as the size and number of primary tumors.In our country, according to the latest statistical report for the year 2004 1 the incidence of bladder carcinoma was 6.7% for male (the fourth most common malignancy) and 2.1% for female population (the ninth most common malignancy), while the Bojanić N, et al.Vojnosanit Pregl 2012; 69(2): 147-150.mortality rate was 4% and 1.6% for male and female population, respectivly.
The change in glucosaminoglycons (GAG) level is interesting in the diagnosis and follow-up of bladder carcinoma.It has been established that GAG is the main constituent of the basic layer of the connective tissue and cellular membrane.Their presence has been evidenced in mucosa and muscular tissue of the urinary bladder [2][3][4] in both healthy bladders and those affected by carcinoma.Their role in antibacterial adherence and formation of urinary stones [5][6][7][8] is already known.All this is suggestive for the potential role that GAG can play in the occurrence of urinary bladder tumors.Changes occurring in the epithelium and deeper structures cases of bladder tumors, suggest that the whole bladder mucosa is preparing for the occurrence of malignancy (primary and recurrent tumor).
The aim of our study was to determine GAG levels in tumor tissue and bladder mucosa of patients with tumor, as well as in healthy mucosa of the bladder, and to compare the results regarding tumor grade and stage as well as in cases of tumor relapse.

Methods
Our study comprised 61 patients with bladder tumor (48 males and 13 females) aged 40-92 years (average age 61.5 years) and 8 controls with healthy bladder mucosa of the comparable age (6 males and 2 females).
The material was sampled by transurethral resection (TUR) in all subjects.Sixty-one tumor biopsies were taken as well as 46 samples of bladder mucosa adjacent to the tumor and no macroscopic signs of abnormalities.Control samples were taken by TUR in 8 patients with no signs of malignancy, but the presence of urinary stones or infections.
The samples were subjected for biochemical analysis as well as for histological analysis.Hematoxylin eosin was used for histological staining.The material taken for biochemical studies was stored at -20°C, and subsequently homogenized at a Potter-Elvehiem's homogenizer at 3000 rpm.The material prepared in this way, both tumorous and mucosal tissues, were studied using the Whitman's method for determination of GAG.The values of GAG were presented in grams of tissue proteins, since proteins are a constant parameter in the tissue.
Tissue GAG levels were determined by the Whitman's method 9 , from a complex with Alcian Blue 8 GX stain (Alcian Blue 8 GX 0.5 g/L in 0.05 mol/L Na-acetate buffer with addition of 0.05 mol/L MgCl 2 , pH 5.8), subsequently dissolved in Na-lauril sulfate.The intensity of color was measured by a spectrophotometer at 620 nm.Tissue proteins were determined with Commasi Brilliant Blue (CBB) stain by spectrophotometry at 595 nm.
The results were processed by nonparametric statistic tests: Mann-Whitney U-test, Kruskall-Wallis test and Wilcoxon test.

Results
Distribution of the patients regarding tumor grade and gender is given is Table 1.
Two patients with grade III tumors entered the study.No signs of invasion were found and they were not particularly analyzed in relation to tumor grade.
According to histopathologic findings and clinical picture, using TNM (Tumor Nodus Metastasis) and World Health Organization (WHO) clasification systems, tumors were classified into three stages (Figure 1).

Fig. 1 -Distribution of patients according to the tumor stage
The biochemical evaluation was done in 57 out of 61 tumor samples (in 4 cases due to techical reasons evaluation was not possible), 46 samples of heatlhy appearing mucosa from urinary bladder with tumor, and 8 samples of healthy bladder mucosa (Table 2).
The values of GAG were expressed in mg GAG/g tissue proteins, in relation to tumor grade in tumor tissue as well as bladder mucosa of patients with tumor (Table 3).
In Table 4 the relationship between GAG values and clinical stages of the tumor is given.
According to individual values of GAG in tumor and mucosal tissue of patients with tumor relapse, no differences in GAG values were noted which might suggest the occurrence of relapse although the values remained within the range obtained elsewhere.
Two year follow-up of tumor relapses according the tumor grade and stage is shown in Table 5.

Discussion
According to the available information on the behavior of superficial bladder carcinoma, it is diffucult to predict further course and occurrence of relapses and outcome of the disease.Not a single tumor marker has been found to have specificity and sensitivity which would make it useful for clinical practice.Therefore, efforts have been focused on detecting a substrate which would satisfy all relevant criteria for a good tumor marker.In addition to the above, we believe that the whole bladder mucosa is influenced by car-cinogens, and that one or more mucosal fields are the vulnerable sites where the absence of protective factors permit neoplasm proliferation.It is difficult to say whether the removed primary, solitary neoplastic lesion still presents a local predisposing factor for the relapse on the same site, or, some other sites, relieved from the protective noxa become new sites of de novo tumor growth since the whole mucosa suffers the influence of the noxious factor.
A decision to evaluate GAG has been made since the available referential data suggested their insufficiently studied role in bladder tumors 10,11 .We focused on qualitative study of GAG intending to identify and possibly, show a correlation of GAG in tumor and mucosa of patients with superficial bladder tumors which might suggest tumor recurrence.The results of de Klerk 2 point out that total GAG levels are mildly reduced in 13 (100%) cases of bladder transitional cell carcinoma with large individual variations.He believes that, due to the variability, total GAG level cannot indicate different grades and stages of tumors.However, our results indicate the opposite: elevation of total GAG values correlates with the increase in tumor stage and grade.All tumors had more elevated GAG values in comparison with healthy mucosa.Moreover, the noted variability in GAG values, in each individual case, remains within the grade and stage of the tumor.The content of particular GAG fractions within the total GAG content cannot be compared since we did not include a qualitative analysis in our study.
The results of our study concerning total GAG content, suggest increased GAG levels in tumorous and mucosal tissue in patients with tumors, as compared with the controls.The difference was statistically significant (p < 0.05).It remains to be answered whether the recorded GAG values result from the discharge of GAG from tumorous tissue, ie preparation of the mucosa for the occurrence of tumor, since the control mucosal tissue does not contain high GAG levels.We recorded elevation of GAG values with higher tumor grade.The lowest values are found in papilloma while the highest were found in cases with tumor grade III.This applies to GAG values in tumorous tissue, while in the same group of patients GAG values in mucosa not affected with tumor is somewhat different.Thus, papillomatous mucosa is characterized with GAG levels below normal, which may re-sult from mucosal stress.Tumor increase GAG levels increase in the surrounding tissue with increase of tumor grade, but not strictly following the tumor grade.It is, nevertheless always increased, and the difference from the controls is statistically significant (p < 0.05).Clinically, GAG levels in tumor tissue rises with the incerase in tumor stage.The lowest increase was noted in Ta stage and it is no statistically significant (p > 0.05) when compared with the controls, while in stages T1 and T2 showed a significant increase.
Mucosa of patients with tumor also shows increased GAG levels in all stages of the disease, and the difference from the controls was statistically significant (p < 0.05).
Out of the 61 patients, relapse was noted in 13 in a 24month period.
Relapse most commonly occurred in patients with grade II tumor, as well as in those with T1 and T2 stages.In none of the analyzed cases with relapse, GAG values in tumor tissue and mucosa deviated from other analyzed cases of the group as a whole, so that the obtained results cannot be used to predict relapse of the disease.

Conclusion
Our study shows that GAG levels rise with the rise in tumor grade and stage in the tumor tissue as well as in surrounding mucosa.Elevation of GAG levels in mucosa of patients with tumor was higher than in tumor itself, suggesting a very important question to be answered.
Whether tumor is an only manifestation of a process occurring in the bladder mucosa with future behavior what is probably already determined remains unclear.