the featUres of expression and prognostic significance of the peptide molecUles connected with the key properties of renal cell carcinoma

Relevance. The modern approach to prognostication of the course of renal carcinoma requires identification of the biological markers, which determine the key properties of the tumor in particular cases. The purpose of this study is to clarify the nature of expression of the molecules connected with the phenotype of the cancer stem cell (CD133, N-cadherin), TGF-α growth factor for tubular epithelium, and carboanhydrase IX (CA-IX) – one of the key molecules connected with VHL by the molecular way of carcinogenesis in cases of renal cancer. Materials and methods. We studied 61 observations of renal cell carcinoma (RCC). The immunohystochemical examination was based on a panel of antibodies, which included antibodies to TGF-α (Аbcam), N-cadgerin (M3613 clone; Dako), CD133 (Prominin-1, Biorbyt), carboanhydrase IX (CAIX; NCL-L-CAIX, Leica Biosystems). The degree of the markers' expression was evaluated by a semiquantitative method depending on the content of the positive-reaction cells in the tumor. The differences between the compared groups were identified through the use of the Mann-Whitney U test and the chi-squared test. Results. Expression of TGF-α was found in 36 observations (59%) of RCC. TGF-α was most frequently present in the papillary (100%) and the clear-cell (55.9%) versions, and only rarely in chromophobic carcinoma (23.1%). A connection was found between the expression level of this marker and the neoplastic differentiation degree (p < 0.05). Expression of N-cadgerin was met in 38 observations (62.3%), CD133 occurred in 37 observations of renal carcinoma (60.6%). N-cadgerin and CD133 were found in the clear-cell and the papillary versions of RCC. There was a connection between the expression of N-cadgerin in the tumor and its invasive potential (p < 0.05). A higher occurrence rate of CD133 expression was recorded in the patients with metastatic forms of RCC, and also in cases of sarcomatoid transformation of the tumor (p < 0.05). Expression of carboanhydrase IX (CA-IX) occurred in 35 observations (57.4%). It was found in the clear-cell version of RCC. In cases of symptomatic RCC, expression of CA-IX was more frequently met (p < 0.05). There was a consistent association between the expression of CA-IX and the tumor size, stage and invasive potential.

Renal cell carcinoma (RCC) remains a significant health burden, accounting for nearly 3% of all malignancies in the adult population.In recent decades, the incidence of RCC has been increasing by 3% to 4% per year.This growth is probably associated with the expansion of methods of intravital kidney imaging, including ultrasound, computed tomography, and magnetic resonance imaging [1].Despite the high prognostic value of the existing diagnostic tools for assessing clinical and histological features of the tumor, its stage, and the degree of differentiation, they cannot accurately predict the course of the disease in a particular patient or the individual characteristics of RCC.Evaluating tumor prognosis at different stages is also challenging.The current approach to predicting the course of RCC involves searching for new biological markers that allow estimation of the most important characteristics of the tumor, such as proliferative activity, resistance to apoptosis, invasion and metastasis ability, and suppression of immune reactions [2].
The aim of this study was to evaluate the expression of molecules associated with cancer stem cell phenotype (CD133, N-cadherin): transforming growth factor (TGF)-α (growth factor for epithelial tubules) and carbonic anhydrase IX (CAIX), one of the key molecules involved in von Hippel-Lindau (VHL)-associated carcinogenesis in RCC.

TGF-α
TGF-α is a multifunctional regulatory peptide with a wide range of effects related to cell growth and differentiation.In the kidney, it stimulates proliferation of proximal tubule epithelial cells.The effects of TGF-α are mediated through epidermal growth factor receptors [3,4].TGF-α expression was observed in 36 RCC patients (59%).A positive TGF-α reaction was more common in clear cell (55.9%) (Fig. 1-3) and type 1 papillary (100%) variants (Fig. 4), whereas it was less common in chromophobe RCC (23.1%) (Fig. 5).Expression of TGF-α was primarily detected in the tumor cell cytoplasm (59%), and less frequently along cell membranes (32.8%).Analyzing the association between TGF-α expression and clinical characteristics of RSS, we found that such factors as age, gender, body mass index, history of hypertension, RCC symptoms, and laboratory signs of chronic kidney disease had no effect on TGF-α expression.We found a correlation between the level of TGF-α production and the degree of tumor differentiation (χ 2 = 6.816; p < 0.05) (Table 1).

N-cadherin
N-cadherin is a membrane protein of the cadherin superfamily that is involved in the formation of calcium-dependent cell-cell contacts.A high level of N-cadherin expression is typical of embryonic mesoderm.Recent studies demonstrated that N-cadherin can be used as a marker of the epithelial-mesenchymal transition, which has a crucial role in increasing tumor aggressiveness (invasion and metastasis) [5].
Expression of N-cadherin was observed in 38 patients (62.3%).It was detected significantly more frequently in clear cell RCC (80%) (p < 0.0001) (Fig. 6) and type 1 papillary RCC (76.9%) (p < 0.0001) (Fig. 7) than original papers in the chromophobe variant, which had no expression of this marker.N-cadherin was primarily located in the cell membrane (75%), and sometimes in the cytoplasm (35.4%).Analyzing possible correlations between the expression of N-cadherin and clinical characteristics of RCC, we found that patients suffering from chronic kidney disease (glomerular filtration rate [GFR] < 60) were more likely to express N-cadherin (χ 2 = 5.67; p < 0.05).We also analyzed the main morphological characteristics of RCC that have a prognostic value (Table 2) and found that N-cadherin production in a tumor was correlated with its invasive potential (χ 2 = 6.486; p < 0.05).

CD133
СD133 (prominin-1) is a transmembrane glycoprotein expressed in hematopoietic stem cells [6].This protein has recently been used as a marker of cancer stem cells.Hypoxia and activation of hypoxia-inducible factor (HIF)-1α can induce proliferation of CD133-positive cells.The level of CD133 production may vary depending on the condition of the surrounding cells [7].
We found an association between the patient`s age and CD133 expression (χ 2 = 17.467; p < 0.01): strong expression was more frequent among young patients.The mean age of patients with strong CD133 expression was 56.64.8 ± 2.21 years, p = 0.014).The correlation was observed for both membranous and cytoplasmic expression.A significant association was found between the level of CD133 production and the risk of decreased GFR in the early postoperative period (χ 2 = 4.155; p < 0.05).The frequency of CD133 expression was higher in individuals with metastatic forms of RCC.Moreover, we found a significant correlation between CD133 expression in tumor cells and the probability of sarcomatoid transformation (χ 2 = 6.480; р < 0.05) (Table 3).

CAIX
CAIX is an HIF-1α-regulated transmembrane protein that is associated with tumor growth, aggressive tumor phenotype, and poor prognosis in clear cell RCC [8].CAIX is believed to be involved in the regulation of intracellular and extracellular pH levels in response to hypoxia in tumor tissue.
CAIX expression was observed in 35 patients (57.4%), primarily in individuals with clear cell RCC (97.1%) (Fig. 11 and 12).One patient with chromophobe RCC had a weak focal reaction with anti-CAIX antibodies; patients with the papillary type of tumor had no CAIX expression.Patients with symptomatic RCC were more likely to have CAIX expression (χ 2 = 6.213; p < 0.05).We also found a significant association between the level of CAIX expression and tumor size (χ 2 = 7.710; p < 0.05), stage (χ 2 = 8.535; p < 0.05), and invasive potential (χ 2 = 5.111; p < 0.05).No correlation between the degree of differentiation and the level of CAIX production was observed (

Таблица 4 Связь между экспрессией CA-IX, морфологическими и прогностическими характеристиками почечноклеточного рака discUssion
Our study demonstrated that the levels of TGF-α, Ncadherin, CD133, and CAIX expression differ among various RCC histological subtypes, which reflects the different molecular pathways underlying their development.The CAIX protein was detected only in clear cell tumors, whereas N-cadherin and CD133 were expressed in both clear cell and type 1 papillary RCC and were absent in chromophobe tumors.Our results are in agreement with previous investigations.Knebelmann et al. (2009) found that TGF-α expression can be sup-pressed by the VHL protein.A number of authors demonstrated increased TGF-α production in clear cell and papillary RCC compared with normal tissue [9,10].They also showed that this protein is likely to be among the factors inducing cancer development in the early stages.The absence of TGF-α in the chromophobe variant of RCC is probably responsible for the low malignant potential of these tumors and their poor vascularization [11]  Histogenetic correlations between these markers and various forms of RCC allow them to be used in morphological differential diagnosis.Furthermore, detection of these proteins in biological fluids (blood, urine) can provide valuable information for screening and monitoring of RCC.
The involvement of the molecules assessed in this study in various molecular pathways of RCC deve-  [20].CD133-positive cells in RCC are assumed to be a source of differentiated malignant cells and support tumor growth [21].
High CAIX expression is associated with favorable prognosis in localized and metastatic RCC [22].The level of CAIX expression is inversely proportional to the risk of metastasis (р = 0.036).Patients with high CAIX expression had better survival, even after adjustment for such factors as T stage, Fuhrman grade, and overall patient condition (р ≤ 0.005) [23].Low CAIX expression (≤85%) was associated with unfavorable outcomes in patients with metastatic RCC (OR3.10;р < 0.001), even after adjustment for clinical and pathological factors (OR4.76;р < 0.001) [24].Ingles et al. (2016) also demonstrated that tumors with low levels of CAIX expression have a worse prognosis (high risk of recurrence and poor cancer-specific and overall survival) [25].
Our findings suggest high prognostic values for TGF-α, N-cadherin, CD133, and CAIX.We found a correlation between TGF-α expression and the degree of tumor differentiation; expression of N-cadherin, CD133, and CAIX; and metastatic potential of the tumor.Thus, the markers can be used for individual prognosis of the disease course as well as for estimating the risk of cancer progression in each particular patient.conclUsion Expression of TGF-α is significantly more common in type 1 papillary and clear cell RCC than in chromophobe cancer.A statistically significant association was found between the level of TGF-α expression and the degree of tumor differentiation.N-cadherin was expressed in type 1 papillary and clear cell RCC, whereas the chromophobe variant had no expression of this marker.A correlation between N-cadherin production and stage 3 and 4 chronic kidney disease was established.Invasive forms of RCC demonstrated N-cadherin expression significantly more frequently.CD133 expression was detected in type 1 papillary and clear cell RCC, whereas the chromophobe variants lacked this marker.CD133-expressing tumor cells are more typical of younger patients.A significant association was found between the level of CD133 expression and the risk of decreased GFR in the early postoperative period.Metastatic forms of RCC are more likely to have CD133-positive tumor cells: there is a correlation between the positivity of the reaction and the probability of sarcomatoid transformation.Expression of CAIX is typical of clear cell RCC.The level of CAIX expression was significantly associated with tumor size, stage,