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UDC 611.611:616.61-006-074 doi.org:10.31684/2541-8475.2019.1(13).25-31
NUCLEOPHOSMIN/B23 ONCOPROTEIN IN THE CLEAR-CELLED RENAL TUMOR: INTERACTION WITH CLINICAL AND ANATOMICAL PROGNOSIS FACTORS
1Altai State Medical University, Barnaul
2Altai branch of the N.N. Blokhin National Medical Research Centre of Oncology, Barnaul
3Institute of Molecular Pathology and Pathomorphology of the Federal Research Centre of Fundamental and
Translational Medicine, Novosibirsk
I.P. Bobrov12, M.N. Myadelets1, I.V. Klimachev1, A.Yu. Dolgatov1, A.V. Lepilov1, T.M. Cherdantseva1, N.G. Kryuchkova1, A.M. Avdalyan12, E.L. Lushnikova3, O.P. Molodykh3
Research objective. To study the nucleophosmin/B23 oncoprotein expression in cells of the clear-celled renal tumor depending on clinical and morphological parameters and postoperative survival of patients. Materials and methods. 83 kidney cancer drugs were studied. Nucleophosmin/B23 expression was revealed by immunohistochemical method, the expression severity was assessed by calculating the integral optical density of the substrate in the cell nuclei.
Results. It is shown that the integral optical density of nucleophosmin/B23 in tumor cells was connected with a number of important prognostic factors: stages of the disease by TNM, the size of the tumor node, the degree of nuclear atypia by Fuhrman, the presence of metastases, and 5 year postoperative survival of patients. Conclusions. The study of the integral optical density of nucleophosmin/B23 can serve as an additional prognosis factor in clear-celled renal tumor.
Key words: clear-celled renal tumor, nucleophosmin/B23, prognosis.
Finding new reliable markers of kidney cancer prognosis is an urgent task of modern oncourology.
According to modern data, nucleophosmin (B23, NPM1, newmatrine, NO38) protein is the main argentophilic protein of the nucleolus [1]. By chemical structure, nucleophosmin is a phosphoprotein. It is found in all mammal cells studied to date, but most of all it is found in tumor cells, where its number increases more than 20 times [2, 3]. The nucleophosmin protein gene is located on chromosome 5 in locus 5q35 [4]. This gene consists of 12 functional domains and encodes several oncoprotein isoforms. In humans, B23 protein exists in the form of two isoforms being the products of alternative splicing of one gene [5]. The dominant isoform is B23.1 (294 amino acid residues, electrophoretic mobility 37-38 kDa), the minor isoform is B23.2 (257 amino acid residues, electrophoretic mobility 35-36 kDa). Isoform B23.1 localizes mainly in the nucleolus, and isoform B23.2 is contained mainly in nucleoplasm [6]. Isoform B23.2 differs from B23.1 by the absence of 35 last amino acids at the C-end of the molecule. Both isoforms function in monomeric (N-end sequence) and oligomeric (C-end sequence) form [7].
In tumor cells, there is hyperexpression of nucleophosmin and formation of specific forms of this protein, some of which are monomers, some are oligomers, and the oligomeric state and distribution of monomeric and oligomeric forms between nucleoli and nucleoplasm change in the process of carcinogenesis [8]. According to some authors, tumor cells contain 5-10 times more isoforms B23.1
compared to isoforms B23.2 [9]. Hyperexpression of B23 protein in tumor cells is also accompanied by the appearance of its abnormal structural variants (shortened, mutant, chimeric) [10]. For example, mutant nucleophosmin is localized in cytoplasm, and chimeric one, as a rule, has nuclear cytoplasmic localization [11].
Nucleophosmin is a polyfunctional protein included in various intracellular processes. The main functions of this protein are: chaperonin activity; ribosom biogenesis stimulation; control of chromosomes centromere duplication; regulation of the cell cycle and apoptosis; proliferation activation and participation in the processes of the cellular response to stress and cell malignization [12].
In the literature, there is a small number of works devoted to the study of nucleophosmin in malignant neoplasms [13, 14, 15, 16, 17, 18, 19, 20]. It is shown that in malignant neoplasms of the liver, prostate, large bowel, urinary bladder, stomach, and salivary gland, nucleophosmin expression increases dramatically. In the literature, we found only one foreign work where nucleophosmin expression in kidney cancer was investigated. Thus, Sari A. et al. (2012) [21] showed that nucleophosmin expression in tumor cells correlated with the degree of nuclear atypia. The authors note that detection of nucleophosmin expression can be a useful immunohistochemical marker for differential diagnosis between oncocytoma and chromophobe kidney cancer.
The research objective is to study the interaction of nucleophosmin/B23 oncoprotein expression
with clinical and anatomical prognosis factors of clear-celled renal tumor and postoperative survival of patients.
Materials and methods
As the material for the study, we used 83 kidneys resected for cancer in the Altai branch of the N.N. Blokhin National Medical Research Centre of Oncology of the Russian Federation, Barnaul. The mean age of patients was 57.6±1.1 years. There were 38 men (45.8%) and 45 women (54.2%).
In tumor grouping, according to clinical stages (I-IV), 46 (55.4%) observations corresponded to stage I (T1N0M0); 10 (12.05%) observations to stage II (T2N0M0); 17 (20.5%) observations to stage III (T1N1M0, T2N1M0, T3N0M0, T3N1M0), and 10 (12.05%) observations to stage IV (T4N0M0, T4N1M0, TanyN2M1, TanyNanyMl). The degree of tumor malignancy was estimated by Fuhrman S.A. The studied material included 35 (42.2%) tumors of GI degree of anaplasia; 21 (25.3%) tumors of GII degree of anaplasia; 17 (20.5%) tumors of GIII degree of anaplasia, and 10 (12%) carcinomas of GIV degree of anaplasia.
There were 15 (18.1%) metastatic carcinomas and 68 (81.9%) localized ones. The mean largest size of the tumor node was 7.1±0.4 cm. Before the surgery, patients did not receive drug treatment.
In the manufacture of histological preparations, the method of tissue matrices was used. In each case, the tumor tissue columns were taken from paraffin blocks (donor blocks) after a preview of the histological preparation with a needle puncher with an internal diameter of 2.0 mm. Then the columns (10-15 pcs.) were placed in paraffin block-recipients 20x20 mm in size. Serial histological sections 4 |jm thick were made from paraffin block-recipients with the help of semi-automatic rotor microtome and transferred to glass (to one glass from two paraffin blocks).
Immunohistochemical detection of nucleophos-min/B23 nucleolar protein was carried out using monoclonal antibodies - nucleophosmin n/B23, clone mouse mAb23 (Labvision firm). The integrated optical density (IOD) of nucleophosmin/B23 in nuclei was estimated using a computer image analysis system consisting of a Leica DME micro-
scope, Leica EC3 digital camera (Leica Microsystems AG, Germany), personal computer, and Vide-oTest-Morphologiia 5.2 software. Nucleophosmin/ B23 IOD values were expressed in relative units (RU). At least 25-30 tumor cells were evaluated.
Statistical processing of the material was carried out with the use of the Statistica 10.0 statistical package. In the case of the normal distribution of data obtained, the methods of parametric statistics (Student t-test) were used while checking statistical hypotheses, and if the obtained data did not meet the criteria of normal distribution (Shapiro-Wilk test W=0.89, p<0.01), the Kolmogorov-Smirnov test or Mann-Whitney U-test was applied. Survival curves were constructed using the Kaplan-Meier method, the statistical significance of survival curves was evaluated using the log-rank test. Correlation relationship were evaluated using the Pearson test. The data were considered reliable at p<0.05.
Results and discussion
In the study of nucleophosmin/B23 oncoprotein expression in tumor cells of clear-cell cancer, the oncoprotein had a nucleolar localization in 100% of cells, while the nuclei were painted brown of varying intensity. In general, regardless of clinical and anatomical parameters of carcinomas, quantitative analysis of nucleophosmin/B23 IOD showed that the median of protein IOP per 1 core was 1023.6±56.7 RU, the margins of fluctuations ranged from 118.9 RU to 15778.3 RU.
In correlation analysis, the connection between nucleophosmin/B23 and the sex of patients was not found (r=0.07; p=0.50) (Table 1). In men, nucleophosmin/B23 protein IOD per 1 core was 1305.8±57.9 RU. In women, protein IOD did not significantly differ from that in men and amounted to 1523.4±94.4 RU (p=0.06) (Table 2).
There was also no correlation between nucleophosmin/B23 protein IOD and the age of patients (r=0.005; p=0.96) (Table 1). In the study of protein depending on the age of patients, the lowest value of nucleophosmin/B23 IOD was noted in the age group from 30 to 39 years old (658.2±56.5 RU), and the highest one in the age from 70 to 79 years (2025.5±273.5 RU) (Table 2).
Table 1
Correlation of nucleophosmin/B23 IOD with prognostic clinical and morphological parameters
Clinical and morphological parameter Correlation coefficient (r) Reliability (p)
Sex of patients 0.07 0.5
Age of patients 0.005 0.96
Clinical stage 0.57 0.0001
Tumor size 0.58 0.0001
Degree of anaplasia by Fuhrman 0.70 0.0001
Presence of metastases 0.40 0.0001
Table 2
Nucleophosmin/B23 integrated optical density depending on clinical and anatomical parameters
Clinical and anatomical parameter Nucleophosmin/B23 IOD (RU) Reliability (p)
Sex: male 1305.8±57.9
female 1523.4±94.4 p=0.06
Age: 30-39 years old (1) 40-49 years old (2) 50-59 years old (3) 60-69 years old (4) 70-79 years old (5) 658.2±56.5 2007.7±197.9 1227.6±59.3 1176.8±82.5 2025.5±273.5 P51=0.005 P5-2=0.6 P53=0.00002 P5-4=0.00009
TNM stage: Stage I Stage II 956.1±31.4 2034.6±134.3 p=0.000001
Stage III Stage IV 2525.9±197.5 3825.9±521.7 p=0.09 p=0.009
Degree of nuclear atypia by Fuhrman: GI GII GIII GIV 728.1±19.2
862.2±24.6 p=0.00002
2166.9±63.2 p=0.001
4711.7±364.7 p=0.0000001
Size of the tumor node:
<7.0 cm 904.2±24.7
>7.0 cm 2555.5±149.2 p=0.001
Metastases: N0 1197.3±43.4
N+ 3098.3±301.0 p=0.0000001
Nucleophosmin/B23 IOD correlated with the clinical stage of the disease (r=0.57; p=0.0001) (Table 1). In tumors at stage I of the pathological process, the average nucleophosmin/B23 protein IOD per 1 core was 956.1±31.4 RU, at stage II - 2034.6±134.3 RU (p=0.0000001), at stage III - 2525.9±197.5 RU (p=0.09), and at stage IV - 3825.9±521.7 RU (p=0.009) (Table 2). Thus, nucleophosmin/B23 protein IOD was interconnected with the clinical stage of the pathological process. At the increase in the stage of the process, the content of nucleophosmin/ B23 protein in tumor cell nuclei also significantly increased.
Nucleophosmin/B23 IOD was associated with the size of the tumor node (r=0.58; p=0.0001) (Table 1). In tumor cells of <7cm in size, IOD was 904.2±24.7 RU, and in tumor nodes of >7 cm, protein IOD significantly increased to 2555.5±149.2 RU per 1 nucleus (p=0.001) (Table 2). Thus, nucleophosmin/ B23 protein IOD was interconnected with the size of the tumor node. With increasing tumor size, nucleophosmin/B23 IOD also increased by 1 nucleus.
Nucleophosmin/B23 IOD correlated with the degree of anaplasia by Fuhrman (r=0.70; p=0.0001) (Table 1). In tumors of the GI degree of anaplasia by Fuhrman, the mean nucleophosmin/ B23 oncoprotein IOD per 1 nucleus amounted to 728.1±19.2 RU, of the GII degree of anaplasia -862.2±24.6 RU (p=0.00002), of the GIII degree of anaplasia - 2166.9±63.2 RU (p=0.001), and the mean
IOD at the GIV degree of anaplasia - 4711.7±364.7 RU (p=0.0000001) (Figure 1, 2) (Table 2). Thus, in the clear-celled renal tumor, nucleophosmin/B23 protein IOD was interconnected with the degree of tumor cell anaplasia. A significant increase in the content of nucleophosmin/B23 protein in cell nuclei was revealed at the increase of tumor anaplasia.
Nucleophosmin/B23 IOD was associated with metastasis of the clear-celled renal tumor (r=0.40; p=0.0001) (Table 1). In cells of localized tumors, nucleophosmin/B23 oncoprotein IOD was 1197.3±43.4 RU per 1 nucleus, and in metastatic tumors, protein IOD significantly increased to 3098.3±301.9 RU per 1 nucleus (p=0.0000001) (Table 2). Thus, in the cell nuclei of metastasizing tumors of the clear-celled renal cancer, a significantly larger nucleophosmin/B23 protein IOD was noted compared to localized tumors.
We conducted a study of 5 year postoperative survival of patients depending on nucleophosmin/ B23 IOD in tumor cells. In the Kaplan-Meyer survival analysis, the graph shows that when nucleophosmin/B23 IOD in tumor cells was <1000 RU, the cumulative share of survivors was 0.98 (98%) by day 1800 after the operation, and when nucleophosmin/B23 IOD in tumor cells was >1000 RU, the cumulative share of survivors was 0.60 (60%). Comparative analysis of data using the logarithmic rank criterion revealed that differences between survival curves in study groups are reliable (log-rank; p=0.01) (Figure 3).
Figure 1. Nucleophosmin/B23 protein expression in tumor cell nuclei of GI degree of anaplasia (shown by arrows): a — coloration of the tumor with haematoxylin and eosin, increase x400; b - immunohistochemical reaction, increase x1000.
Figure 2. Nucleophosmin/B23 protein expression in tumor cell nuclei of GIV degree of anaplasia (shown by arrows): a — coloration of the tumor with haematoxylin and eosin, increase x400; b - immunohistochemical reaction, increase x1000.
Figure 3. Cumulative ratio of 5 year survival in patients with RCC depending on nucleophosmin/B23 IOD in tumor cells.
Conclusion
Summarizing the abovementioned, we can conclude that nucleophosmin/B23 nucleolar oncoprotein expression in the clear-celled renal tumor was interconnected with clinical and morphological prognostic parameters of carcinomas and 5 year postoperative survival of patients. With the increase in the clinical stage, the tumor node size, the degree of anaplasia, and the presence of nucleop-hosmin/B23 protein IOD metastases significantly increased per 1 nucleus. In our opinion, the increase in nucleophosmin/B23 IOD significantly increases the ability of tumor cells to invasive growth and metastasis. Therefore, finding populations of tumor cells with high nucleophosmin/B23 IOD per 1 nucleus in the clear-celled renal tumor may indicate the risk of metastases and unfavourable prognosis for the course of the disease. Determination of nucleophosmin/B23 protein IOD in tumor cells of primary carcinoma can be used in assessing the risk of metastases at the preoperative stage, predicting the course of RCC, and planning the scope of surgical treatment. Nucleophosmin/B23 protein IOD assessment in the clear-celled renal tumor can serve as an additional marker in determining the disease prognosis in combination with traditional classical prognostic factors.
Conflict of interest. The authors declare that there is no conflict of interest.
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Contacts
Corresponding author: Bobrov Igor Petrovich, Senior Researcher of the Morphological Laboratory of the Center for Medical and Biological Research, Altai State Medical University, Barnaul. 656038, Barnaul, Lenina Prospekt, 40. Tel.: (3852) 408439. E-mail: ig.bobrov2010@yandex.ru
Author information
Myadelets Mikhail Nikolaevich, pathologist, Postgraduate student of the Department of Forensic Medicine and Pathological Anatomy named after professor V.N. Kryukov with the Course of FVE, Altai State Medical University, Barnaul. 656038, Barnaul, Lenina Prospekt, 40. Tel.: (3852) 408439. E-mail: mic7818@yandex.ru
Klimachev Ilya Vladimirovich, pathologist, Postgraduate student of the Department of Forensic Medicine and Pathological Anatomy named after professor V.N. Kryukov with the Course of FVE, Altai State Medical University, Barnaul. 656038, Barnaul, Lenina Prospekt, 40. Tel.: (3852) 408439. E-mail: ko222@yandex.ru
Dolgatov Andrey Yuryevich, Candidate of Medical Sciences, Associate Professor of the Department of Forensic Medicine and Pathological Anatomy named after professor V.N. Kryukov with the Course of FVE, Altai State Medical University, Barnaul.
656038, Barnaul, Lenina Prospekt, 40.
Tel.: (3852) 408439.
E-mail: adolgatov@yandex.ru
Lepilov Aleksandr Vasilyevich, Doctor of Medical Sciences, Head of the Department of Forensic Medicine and Pathological Anatomy named after professor V.N. Kryukov with the Course of FVE, Altai State Medical University, Barnaul. 656038, Barnaul, Lenina Prospekt, 40. Tel.: (3852) 408439. E-mail: lepilov@list.ru
Cherdantseva Tatyana Mikhailovna, Doctor of Medical Sciences, Professor of the Department of Oncology and Radiation Diagnostics with the course of FVE, Altai State Medical University, Barnaul.
656038, Barnaul, Lenina Prospekt, 40.
Tel.: (3852) 408439.
E-mail: cherdan.morf@yandex.ru
Kryuchkova Natalia Gennadyevna, Assistant Professor of the Department of Forensic Medicine and Pathological Anatomy named after professor V.N. Kryukov with the Course of FVE, Altai State Medical University, Barnaul. 656038, Barnaul, Lenina Prospekt, 40. Tel.: (3852) 408439. E-mail: cruckova@yandex.ru
Avdalyan Ashot Meruzhanovich, Doctor of Medical Sciences, Head of the Laboratory for Research of Molecular Genetic Characteristics of Tumors, Altai branch of the N.N. Blokhin National Medical Research Centre of Oncology, Barnaul. 656049, Barnaul, ul. Nikitina, 77. Tel.: +7 (3852) 507399. E-mail: ashot_avdalyan@mail.ru
Lushnikova Elena Leonidovna, Doctor of Biological Sciences, Professor, Academician of the Russian Academy of Sciences, Director of the Institute of Molecular Pathology and Pathomorphology of the Federal Research Centre of Fundamental and Translational Medicine, Head of the Department of Molecular Cell Biology and Morphology, Head of the Laboratory of Cytology and Cell Biology, Novosibirsk.
639117, Novosibirsk, ul. Timakova, 2. Tel.: +7 (383) 3348003. E-mail: pathol@inbox.ru
Molodykh Olga Pavlovna, Doctor of Biological Sciences, Head of the Laboratory of Mechanisms of Pathological Processes of the Institute of Molecular Pathology and Pathomorphology of the Federal Research Centre of Fundamental and Translational Medicine, Novosibirsk. 639117, Novosibirsk, ul. Timakova, 2. Tel.: +7 (383) 3348003. E-mail: pathol@inbox.ru
