The features of expression and prognostic significance of the peptide molecules connected with the key properties of renal cell carcinoma

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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.

The study included 61 patients with RCC (35 cases of clear cell RCC, 13 cases of type 1 papillary RCC, and 13 cases of chromophobe RCC). We performed morphological examination of tissue samples collected during radical nephrectomy. The specimens were fixed in 10% buffered neutral formalin and embedded in paraffin according to standard procedures. Histological slides were stained with hematoxylin and eosin. The following antibodies were used for immunohistochemical analysis: anti-TGF-α antibodies (TGF-α, dilution 1: 100; Аbcam, Cambridge, United Kingdom), anti-N-cadherin antibodies (N-cadherin, клон M3613; dilution 1: 50; Dako, Glostrup, Denmark), anti-CD133 antibodies (prominin-1 dilution 1: 200; Biorbyt, Cambridge, United Kingdom), and anti-CAIX antibodies (CAIX; NCL–L-CAIX, dilution 1: 100; Leica Biosystems, Hamburg, Germany). A semiquantitative method was used to estimate the expression of immunohistochemical markers: 0–no expression (in 0% of tumor cells), 1–weak expression (in up to 10% of tumor cells), 2–moderate expression (in 11%–30% of tumor cells), and 3–strong expression (in over 30% of tumor cells). Additionally, we evaluated the location of positive staining as membranous, cytoplasmic, or nuclear. Statistical analysis was performed with the Statistica 6.1 software package. Fisher’s exact test and Pearson’s χ2 test were used to assess differences between binary variables; continuous variables were compared by the Mann–Whitney U test.

Results

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 (χ2 = 6.816; p < 0.05) (Table 1).

 

Fig. 1. Moderately differentiated renal cell carcinoma (RCC) (clear cell variant), nuclear grade 2. Pronounced focal positive cytoplasmic reaction with anti-transforming growth factor (TGF)-α antibodies

 

Fig. 2. Moderately differentiated renal cell carcinoma (RCC) (clear cell variant), nuclear grade 2–3. Pronounced diffuse positive cytoplasmic reaction with anti-transforming growth factor (TGF)-α antibodies

 

Fig. 3. Poorly differentiated renal cell carcinoma (RCC) (clear cell variant) with sarcomatoid transformation. Pronounced diffuse positive cytoplasmic reaction anti-transforming growth factor (TGF)-α antibodies

 

Fig. 4. Highly differentiated renal cell carcinoma (RCC) (type 1 papillary variant). Diffuse positive cytoplasmic and membranous reaction with anti-transforming growth factor (TGF)-α antibodies

 

Fig. 5. Moderately differentiated renal cell carcinoma (RCC) (chromophobe variant). Focal positive cytoplasmic reaction with anti-transforming growth factor (TGF)-α antibodies

 

Table 1. Correlation between TGF-α expression and morphological and prognostic characteristics of RCC

Таблица 1. Связь между экспрессией TGF-α, морфологическими и прогностическими характеристиками почечно-клеточного рака

Characteristic	Histological type						Total	χ2
	Clear cell		Type 1 papillary		Chromophobe
	No expression detected	Expression detected	No expression detected	Expression detected	No expression detected	Expression detected
Size of the primary tumor
<4 cm	2	3	0	8	3	1	17
4–7 cm	6	9	0	3	3	2	23
>7 cm	7	8	0	2	4	0	21
Total	15	20	0	13	10	3	61	2.399 p > 0.05
Tumor stage (T)
Т1а	2	6	0	8	3	1	20
Т1b	1	1	0	2	1	1	6
Т2	0	0	0	0	1	0	1
Т3а	11	10	0	3	4	1	29
Т3b	1	3	0	0	1	0	5
Total	15	20	0	13	10	3	61	5.083 p > 0.05
Invasion into the tissue and blood vessels of the renal sinus, renal vein, and its branches
Yes	11	10	0	3	4	1	29
No	4	10	0	10	6	2	32
Total	15	20	0	13	10	3	61	2.637 p > 0.05
Presence of regional lymphogenous metastases
Yes	1	0	0	0	0	0	1
No	14	20	0	13	10	3	60
Total	15	20	0	13	10	3	61	1.464 p > 0.05
Characteristic	Histological type						Total	χ2
	Clear cell		Type 1 papillary		Chromophobe
	No expression detected	Expression detected	No expression detected	Expression detected	No expression detected	Expression detected
No	13	19	0	11	10	3	56
Total	15	20	0	13	10	3	61	0.002 p > 0.05
Degree of differentiation (Fuhrman's nuclear grading)
G1 (grade 1–2)	1	6	0	6	1	1	15
G2 (grade 2–3)	11	11	0	4	8	2	36
G3 (grade 3–4)	3	3	0	3	1	0	10
Total	15	20	0	13	10	3	61	6.816 p < 0.05
Presence of sarcomatoid and rhabdoid transformation
Yes	3	3	0	2	1	0	9
No	12	17	0	11	9	3	52
Total	15	20	0	13	10	3	61	0.052 p > 0.05
Presence of coagulative necrosis foci in the tumor
Yes	2	1	0	4	1	0	8
No	13	19	0	9	9	3	53
Total	15	20	0	13	10	3	61	0.024 p > 0.05

 

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 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 (χ2 = 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 (χ2 = 6.486; p < 0.05).

 

Fig. 6. Poorly differentiated renal cell carcinoma (RCC) (clear cell variant), nuclear grade 3. Pronounced diffuse positive membranous reaction with anti-N-cadherin antibodies

 

Fig. 7. Moderately differentiated renal cell carcinoma (RCC) (type 1 papillary variant), nuclear grade 2. Diffuse positive membranous reaction with anti-N-cadherin antibodies

 

Table 2. Correlation between N-cadherin expression and morphological and prognostic characteristics of RCC

Таблица 2. Связь между экспрессией N-кадгерина, морфологическими и прогностическими характеристиками почечно-клеточного рака

Characteristic	Histological type						Total	χ2
	Clear cell		Type 1 papillary		Chromophobe
	No expression detected	Expression detected	No expression detected	Expression detected	No expression detected	Expression detected
Size of the primary tumor
<4 cm	1	3	3	6	4	0	17
4–7 cm	3	11	0	3	6	0	23
>7 cm	3	14	0	1	3	0	21
Total	7	28	3	10	13	0	61	3.52 p > 0.05
Tumor stage (Т)
T1a	1	7	3	5	4	0	20
Т1b	0	1	0	2	1	0	4
Т2	0	0	0	0	0	0	0
Т3а	5	17	0	3	6	0	31
Т3b	1	3	0	0	2	0	6
Total	7	28	3	10	13	0	61	1.777 p > 0.05
Invasion into the tissue and blood vessels of the renal sinus, renal vein, and its branches
Yes	1	12	0	7	3	0	23
No	6	16	3	3	10	0	38
Total	7	28	0	10	13	0	61	6.486 p < 0.05
Presence of regional lymphogenous metastases
Yes	0	1	0	0	0	0	1
No	7	27	3	10	13	0	60
Total	7	28	3	10	13	0	61	0.496 p > 0.05
Presence of distant lymphogenous and hematogenous metastases
Yes	1	3	0	2	0	0	6
No	6	25	3	8	13	0	55
Total	7	28	3	10	13	0	61	0.224 p > 0.05
Degree of differentiation (Fuhrman's nuclear grading)
G1 (grade 1–2)	1	7	2	4	1	0	15
G2 (grade 2–3)	6	19	1	3	11	0	40
G3 (grade 3–4)	0	2	0	3	1	0	6
Total	7	28	3	10	13	0	61	2.815 p > 0.05
Presence of sarcomatoid and rhabdoid transformation
Yes	2	4	0	2	1	0	9
No	5	24	3	8	12	0	52
Total	7	28	3	10	13	0	61	0.086 p > 0.05
Presence of coagulative necrosis foci in the tumor
Yes	1	2	0	4	1	0	9
No	6	26	3	6	12	0	52
Total	7	28	3	10	13	0	61	0.086 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].

CD133 was expressed in 37 patients (60.6%). CD133-positive cells were more frequently detected in clear cell RCC (80%) (Fig. 8 and 9) and papillary RCC (69.2%) (Fig. 10), whereas patients with chromophobe cancer had no CD133 expression. The marker was primarily located in the cytoplasm (60.6%), and less frequently in the cell membrane (44.3%).

 

Fig. 8. Moderately differentiated renal cell carcinoma (RCC) (clear cell variant), nuclear grade 2. Pronounced focal positive cytoplasmic reaction with anti-CD133-antibodies

 

Fig. 9. Poorly differentiated renal cell carcinoma (RCC) (clear cell variant) with sarcomatoid transformation, nuclear grade 3. Pronounced positive cytoplasmic reaction with anti-CD133 antibodies

 

Fig. 10. Moderately differentiated renal cell carcinoma (RCC) (type 1 papillary variant), nuclear grade 2–3. Pronounced positive cytoplasmic reaction with anti-CD133 antibodies

 

We found an association between the patient`s age and CD133 expression (χ2 = 17.467; p < 0.01): strong expression was more frequent among young patients. The mean age of patients with strong CD133 expression was 56.3 ± 5.4 years, whereas patients with no CD133 expression were significantly older (mean age 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 (χ2 = 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 (χ2 = 6.480; р < 0.05) (Table 3).

 

Table 3. Correlation between CD133 expression and morphological and prognostic characteristics of RCC

Таблица 3. Связь между экспрессией CD133, морфологическими и прогностическими характеристиками почечно-клеточного рака

Characteristic	Histological type						Total	c2
	Clear cell		Type 1 papillary		Chromophobe
	No expression detected	Expression detected	No expression detected	Expression detected	No expression detected	Expression detected
Size of the primary tumor
<4 cm	1	4	3	5	4	0	17
4–7 cm	5	10	0	3	5	0	23
>7 cm	1	14	1	1	4	0	21
Total	7	28	4	9	13	0	61	5.810 p > 0.05
Tumor stage (Т)
T1a	1	7	3	5	4	0	20
Т1b	0	2	0	2	2	0	6
Т2	0	0	0	0	1	0	1
Т3а	5	16	1	2	6	0	30
Т3b	1	3	0	0	0	0	4
Total	7	28	4	9	13	0	61	0.390 p > 0.05
Invasion into the tissue and blood vessels of the renal sinus, renal vein, and its branches
Yes	5	16	1	2	5	0	29
No	2	12	3	7	8	0	32
Total	7	28	4	9	13	0	61	0.046 p > 0.05
Presence of regional lymphogenous metastases
Yes	1	0	0	0	0	0	1
No	6	28	4	9	13	0	60
Total	7	28	4	9	13	0	61	1.567 p > 0.05
Characteristic	Histological type						Total	c2
	Clear cell		Type 1 papillary		Chromophobe
	No expression detected	Expression detected	No expression detected	Expression detected	No expression detected	Expression detected
No	5	28	2	9	13	0	57
Total	7	28	4	9	13	0	61	6.599 P < 0.05
Degree of differentiation (Fuhrman's nuclear grading)
G1 (grade 1–2)	1	7	1	5	1	0	15
G2 (grade 2–3)	5	16	2	2	11	0	36
G3 (grade 3–4)	1	5	1	2	1	0	10
Total	7	28	4	9	13	0	61	4.431 p > 0.05
Presence of sarcomatoid and rhabdoid transformation
Yes	1	5	1	1	1	0	9
No	6	23	3	8	12	0	52
Total	7	28	4	9	13	0	61	6.480 p < 0.05
Presence of coagulative necrosis foci in the tumor
Yes	1	2	2	2	1	0	8
No	6	26	2	7	12	0	53
Total	7	28	4	9	13	0	61	0.438 p > 0.05

 

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 (χ2 = 6.213; p < 0.05). We also found a significant association between the level of CAIX expression and tumor size (χ2 = 7.710; p < 0.05), stage (χ2 = 8.535; p < 0.05), and invasive potential (χ2 = 5.111; p < 0.05). No correlation between the degree of differentiation and the level of CAIX production was observed (Table 4).

 

Fig. 11. Moderately differentiated renal cell carcinoma (RCC) (clear cell variant, usual structure), nuclear grade 1–2. Pronounced positive membranous reaction with anti-carbonic anhydrase IX (CAIX) antibodies

 

Fig. 12. Moderately differentiated renal cell carcinoma (RCC) (clear cell variant, tubulopapillary structure), nuclear grade 1–2. Positive membranous and cytoplasmic reaction with anti-carbonic anhydrase-IX (CAIX) antibodies

 

Table 4. Correlation between CAIX expression and morphological and prognostic characteristics of RCC

Таблица 4. Связь между экспрессией CA-IX, морфологическими и прогностическими характеристиками почечно-клеточного рака

Characteristic	Histological type						Total	c2
	Clear cell		Type 1 papillary		Chromophobe
	No expression detected	Expression detected	No expression detected	Expression detected	No expression detected	Expression detected
Size of the primary tumor
<4 cm	0	5	4	0	8	0	17
4–7 cm	1	14	3	0	4	1	23
>7 cm	0	15	6	0	0	0	21
Total	1	34	13	0	12	1	61	7.710 p < 0.05
Characteristic	Histological type						Total	c2
	Clear cell		Type 1 papillary		Chromophobe
	No expression detected	Expression detected	No expression detected	Expression detected	No expression detected	Expression detected
Tumor stage (Т)
Т1b	0	2	2	0	2	0	6
Т2	0	0	0	0	1	0	1
Т3а	0	22	3	0	5	1	31
Т3b	1	2	0	0	0	0	3
Total	1	34	13	0	12	1	61	8.535 p < 0.05
Invasion into the tissue and blood vessels of the renal sinus, renal vein, and its branches
Yes	0	21	3	0	5	0	29
No	1	13	10	0	7	1	32
Total	1	34	13	0	12	1	61	5.111 p < 0.05
Presence of regional lymphogenous metastases
Yes	0	1	0	0	0	0	1
No	1	33	13	0	12	1	60
Total	1	34	13	0	12	1	61	0.755 p > 0.05
Presence of distant lymphogenous and hematogenous metastases
Yes	0	3	2	0	0	0	5
No	1	31	11	0	12	1	56
Total	1	34	13	0	12	1	61	0.755 p > 0.05
Degree of differentiation (Fuhrman's nuclear grading)
G1 (grade 1–2)	0	8	6	0	1	0	15
G2 (grade 2–3)	1	20	4	0	10	1	36
G3 (grade 3–4)	0	6	3	0	1	0	10
Total	1	34	13	0	12	1	61	0.142 p > 0.05
Presence of sarcomatoid and rhabdoid transformation
Yes	0	6	2	0	1	0	9
No	1	28	11	0	11	1	52
Total	1	34	13	0	12	0	61	0.373 p > 0.05
Presence of coagulative necrosis foci in the tumor
Yes	0	3	4	0	1	0	8
No	1	31	9	0	11	1	53
Total	1	34	13	0	12	1	61	1.488 p > 0.05

 

Discussion

Our study demonstrated that the levels of TGF-α, N-cadherin, 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 suppressed 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]. Expression of N-cadherin in clear cell RCC was studied by Markovic-Lipkovski et al. (2001), Shimazui et al. (2006), and Behnes et al. (2012) [12–14]. 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 development and progression determines their prognostic value. N-cadherin was shown to be involved in transendothelial migration of cancer cells, which is an important step in metastasis [5]. Increased production of this marker is associated with a poor prognosis of RCC [12, 15, 16].

Costa et al. (2012) demonstrated that nearly 50% of RCCs have CD133 expression. Zhang et al. (2013) observed CD133 expression in 21% of RCC cases. Some authors suggest that high CD133 expression can be a favorable prognostic factor [16–19]. However, Feng et al. (2014) found that patients with an increased serum level of CD133 mRNA have a high risk of recurrence and metastasis [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, and invasive potential. Patients with symptomatic RCC were more likely to express CAIX than those without symptomatic RCC.

About the authors

Dmitrij G. Pasechnik

Rostov State Medical University

Author for correspondence.
Email: dept_kogan@mail.ru

candidate of medical science, associate professor of department of pathological anatomy

Russian Federation, Rostov-on-Don

Mickhail I. Kogan

Rostov State Medical University

Email: dept_kogan@mail.ru

д-р мед. наук, профессор, заведующий кафедрой урологии и репродуктивного здоровья человека с курсом детской урологии-андрологии

Russian Federation, Rostov-on-Don

Zalimhan M. Akhokhov

Emergency Aid City Hospital

Email: dr.ahohov@mail.ru

urologist

Russian Federation, Taganrog

References

Supplementary files