Significance of gene polymorphysm in development of colorectal cancer

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Abstract


Aim. To determine the significance of polymorphism of MTHFR (Ala222Val), XPD (Lis751Gln), XRCC1 (Arg194Trp), XRCC1 (Arg399Gln), XRCC1 (Arg208His), APE1 (Asp148Glu), hOGG1 (ser326Ces), P53 (Pro47Ser), VEGF (C654G), EGFR(A2073T), TNF(G308A), CHEK2 (Ile157Thr), MMP1 (1607 1G>2G), TIMP1(C53CT) genes in development of colorectal cancer.

Materials and Methods. 106 Cases of colorectal cancer in patients who were on treatment in Ryazan Clinical Oncological Dispensary (Ryazan) were analyzed. Genotyping in all patients was performed using the method of isolation of DNA from leukocytes of venous blood with subsequent polymerase chain reaction (PCR) with electrophoretic detection of the result.

Results. No interrelation between the age of patients and polymorphism of any studied gene was recorded at the moment of verification of the diagnosis (р>0.05). Statistically significant relationship was identified between polymorphism of TNF (G308A) gene and the stage of cancer: its homozygous major genotype G/G more commonly occurred in the group of patients with III-IV stage (р=0.047). In the presence of allele of G/G TNF (G308A) gene together with homozygous mutant allele of MMP1 (1607 1G/2G) gene, a direct relationship with increase in the number of patients diagnosed with III-IV stage was noted. This combination of two polymorphisms showed a statistically significant difference in the studied groups (р=0.025). In 8 out of 10 patients with IV stage, the presence of G/G polymorphism in VEGF (C654G) gene was noted. This mutant homozygous variant was much more rare in patents with I (37.5%), II (40%) or III stages (37.5%) (р=0.0147).

Conclusions. The studied genes do not influence the age of manifestation of colorectal cancer and occur at the same frequency in patients of both genders irrespective of the age group. Localization and the extent of differentiation of the tumor do not depend on polymorphism of the studied genes either. The presence of G/A polymorphism of TNF (G308A) gene should be considered a favorable criterion associated with lower aggressiveness of the tumor (р<0.05), whereas identification of the major G/G genotype especially in combination with homozygous mutant allele of MMP1 (1607 1G/2G) gene is an unfavorable factor (р<0.05). The presence of G/G mutant genotype of VEGF (C654G) gene may directly correlate with rapid progression of tumor and with active metastatic spreading (р<0.05).


Full Text

Colorectal cancer (CRC) is the third most common type of cancer in Russia. Annually 60,000 cases of CRC are newly diagnosed. In total, in our country more than 350,000 patients with this pathology are under regular medical check-up.

With this, the results of treatment of this category of patients are unsatisfactory in a large extent due to advanced stage of the process at the moment of verification of the diagnosis. Late identification of CRC (III-IV stages) is the result of absence of screening programs of diagnosis, insufficient knowledge of the causes and risk factors for initiation of this pathology, and peculiarities of the course of the disease that in some cases may be very rapid.

Currently, CRC is treated with combined or complex approach using surgical, radiation and medicinal methods [1]. 

Peculiarities of initiation of cancer, response to the conducted treatment may be largely determined by the individual patient’s genotype that codes for mechanisms that both participate in the carcinogenesis and oppose it [2,3]. In this context, many scientific works are devoted to identification of the connection of polymorphism of genes with pathological processes [4,5] and with different localizations of the tumor [6,7].

The aim of work was to determine the significance of polymorphism of MTHFR (Ala222Val), XPD (Lis751Gln), XRCC1 (Arg194Trp), XRCC1 (Arg399Gln), XRCC1 (Arg208His), APE1 (Asp148Glu), hOGG1 (ser326Ces), P53 (Pro47Ser), VEGF (C654G), EGFR(A2073T), TNF(G308A), CHEK2 (Ile157Thr), MMP1 (1607 1G>2G), TIMP1(C53CT) genes in development of colorectal cancer.

In accordance with the set aim, interrelation of polymorphism with gender, age of patients, localization of tumor process; the degree of tumor differentiation, stage of the disease, depth of invasion and metastatic spread of the tumor was evaluated.

Materials and Methods

The study was carried out on the base of RyazSMU and Ryazan Regional Clinical Oncological Dispensary and was approved by Local Ethic Committee (Protocol №5 of 06.11.15). The patients’ data were used in the work after signing of Informed consent.

Genotyping for polymorphism of the following genes was conducted: MTHFR (Ala222Val), XPD (Lis751Gln), XRCC1 (Arg194Trp), XRCC1 (Arg399Gln), XRCC1 (Arg208His), APE1 (Asp148Glu), hOGG1 (ser326Ces), P53 (Pro47Ser), VEGF (C654G), EGFR (A2073T), TNF (G308A), CHEK2 (Ile157Thr), MMP1 (1607 1G>2G), TIMP1 (C53CT).

Genotyping was conducted in the Central Research Laboratory of Ryazan State Medical University by isolation of DNA from leukocytes of venous blood of the examined patients with subsequent PCR with electrophoretic detection of the result using SNP-EXPRESS» (SPC «Litech», Russia).

The study involved 106 patients receiving treatment for colorectal cancer. The gender composition of the examined patients was approximately equal: 58 men (55%) and 48 women (45%). By the age the patients were divided to 3 groups according to the full amount of years at the moment of manifestation of the disease. I group included patients with relatively early manifestation – before 55 years (the mean age 45.7 years – 24 patients), II - from 55 to 65 years (60.1 – 48 patients) and III group above 65 years (71.6 years – 43 patients). The mean age of all patients at the moment of diagnosis was 61.5 years.

76 Patients (72%) had the diagnosis of colorectal cancer and in 30 patients (28%) tumors were localized in other parts of the large intestine.

The diagnosis was verified in 100% of patients. Histologically, all tumors were represented by adenocarcinomas, mostly with moderate differentiation (G-2, 81%).

The stage of the disease was determined according to the International TNM classification of 8th revision (2018). By the extent of spread of the process, a share of patients with I-II stage (without metastatic lesion of regional lymph nodes) was 45.3% (48 patients), with III stage – also 45.3% (48 patients), and 10 patients (9.6%) had IV stage at the moment of making diagnosis.

Statistical processing of the results was conducted using Excel 2007 (MS, USA), Statistica 10.0 (Stat Soft Inc., USA) software package and online calculators (for Hardy-Weinberg equilibrium – https://wpcalc.com/ en/equilibrium-hardy-weinberg/). Description and comparison of differences in frequencies of qualitative characteristics in the independent groups were based on Pearson chi-squared test, with Yates correction if the quantity of expected events was from 5 to 9, or with Fischer exact test if the quantity was less than 5. The prognostic significance of the characteristic was evaluated with determination of relative risk (RR) with 95% confidence interval (CI). In groups of patients deviation of allele frequency from Hardy-Weinberg equilibrium was also evaluated. In the analysis, statistically significant were considered differences at р<0.05.

Results and Discussion

No statistically significant gender differrences were found in the population of individuals of male and female gender (р>0.05). No interrelation was established between the age of patients and polymorphism of a single studied gene in the groups at the moment of verification of the diagnosis (р>0.05). Besides, in all the age groups Hardy-Weinberg equilibrium was preserved which shows reliability of distribution of allele frequency inside age population groups.

In the analysis of relationship between polymorphism of the studied genes and localization of the tumor process, no statistically significant differences were recorded (р>0.05), as well as in the evaluation of the influence of polymorphism of the studied genes on the extent of tumor differentiation (р>0.05).

The relationship with the stage of tumor process at the moment of diagnosis was evaluated. For the majority of polymorphic genes no direct relationship with increase in stage was noted (р>0.05). A statistically significant relationship was found for polymorphism of TNF G308A gene coding for tumor necrosis factor: its homozygous major genotype G/G was more common in the group of patients with newly identified III-IV stage of the disease (in 69% of patients) in comparison with the group of patients diagnosed at I-II stage (40% of patients, χ2=3.96, р=0.047, RR=1.45 [1.0; 2.03], Table 1).

 

Table 1 Relationship of Polymorphism of TNF and MMP1 Genes with Stage of Disease

Genotype/allele

I-II stage

III-IV stage

χ2, р

RR [95% CI]

TNF(G308A)

G/G

18

33

3.96,

р=0.047

1.45

[1.0;2.03]

G/A

30

25

A/A

0

0

-

-

G

66

91

2.57,

p=0.11

0.77

[0.57;1.04]

A

30

25

TNF(G308A) + MMP1 (1607 2G/2G)

G/G

3

16

5.04,

p=0.025

1.85

[1.13;3.05]

G/A

12

10

 

Besides, relationship of polymorphism of the given gene with other genes was analyzed. It was found that in the coexistence of G/G TNF (G308A) allele with homozygous mutant allele of MMP1 (1607 1G/2G) gene a direct relationship was noted with increase in the number of patients who were first diagnosed at III-IV stage. Proteins encoded by these genes are similar in their biological role and interrelated in terms of potentiating each other. This combination of two polymorphisms showed a statistically significant difference in the studied groups (χ2=5.04, р=0.025, RR=1.85 [1.13; 3.05], Table 1).

Despite a small share of patients with IV stage of the disease included into the study, in 8 of 10 patients of this group G/G polymorphism in VEGF (C654G) gene was noted. This mutant homozygous variant was relatively rare in patients with I (37.5%), II (40%) or III stages (37.5%) (р=0.0147, RR=5.42 [1.21; 24.32], Table 2).

 

Table 2 Stage of Tumor and Polymorphism of VEGF Gene

Genotype/Allele

I-II Stage

III-IV Stage

р

RR [95% CI]

VEGF (C654G)

C/C

10

1

р=0.0147

5.42

[1.21;24.30]

C/G

49

1

G/G

37

8

C

69

3

р=0.1

2.90

[0.88;9.62]

G

123

17

 

On the basis of frequency of genetic polymorphism, no differences in dependence on age of the patient at the moment of manifestation of CRC were revealed. It is quite possible that development of cancer at younger age has genetic substantiation, but it was not shown in the given study.

Localization and the extent of differeniation of the tumor neither depend on polymorphism of the studied genes.

Polymorphic genes MTHFR (Ala222Val), XPD (Lis751Gln), XRCC1 (Arg194Trp), XRCC1 (Arg399Gln), XRCC1 (Arg208His), APE1 (Asp148Glu), P53 (Pro47Ser), EGFR (A2073T), CHEK2 (Ile157Thr), TIMP1 (C53CT) did not show any influence on the tendency of the tumor to more aggressive invasive growth and to metastatic spreading.

With this, a direct relationship was revealed between carriage of major homozygote of TNF (G308A) G/G gene and a high capacity of the tumor to rapid invasion manifested in identification of the disease already in III-IV stage. Carriers of at least one mutant allele of TNF (G308A) gene probably have a weakened function of this cytokine, and, accordingly, its reduced biological influence. These data confirm the known relationship between the activity of cytokine TNF-α and tumor progression due to its ability to enhance cell proliferation, stimulate neoangiogenesis and also to potentiate other factors of inflammation accelerating growth of tumor [8]. Identification of heterozygous condition of the given gene (G/A) may be of favorable prognostic significance due to a longer time required by the tumor for lymphogenic and remote metastasis.

It was also noted that the presence of mutant homozygous allele MMP1 (1607 2G/2G), besides TNF (G308A) G/G genotype, leads to a more aggressive and rapid growth of tumor and is a poor prognostic criterion. This is probably associated with excessive inducing influence of TNF-α cytokine on matrix metalloproteases coded for by MMP1 gene which, in turn, promote early metastatic spread of tumor [9].

Mutation of vascular endothelial growth factor VEGF is a reliable aggressive risk factor of rapid progression of tumor. In our study we did not qualitatively determined expression of this gene in tumor cells, however, it is quite possible that mutant genotype of this allele (G/G) is associated with enhanced growth of vessels and rapid progression of tumor. A small amount of patients with IV stage of tumor process involved into the study did not permit a unambiguous interpretation of the obtained results, but the revealed sharp increase in the mutation of the given gene in this group requires further investigation and probably an additional immune-histochemical examination of tumorous tissues of these patients. 

Conclusion

The significance of polymorphism of MTHFR (Ala222Val), XPD (Lis751Gln), XRCC1 (Arg194Trp), XRCC1 (Arg399Gln), XRCC1 (Arg208His), APE1 (Asp148Glu), hOGG1 (ser326Ces), P53 (Pro47Ser), VEGF (C654G), EGFR (A2073T), TNF(G308A), CHEK2 (Ile157Thr), MMP1 (1607 1G/2G), TIMP1 (C53CT) genes in development of colorectal cancer was analyzed.

The studied genes were equally common in both genders irrespective of the age and did not produce any influence on the age criterion for manifestation of colorectal cancer, neither on localization and the extent of differentiation of tumor.

A reliable connection between polymorphism of TNF (G308A) gene and a tendency of the tumor to rapid invasion into the deeper lying tissues was established that was reflected in identification of the disease at III-IV stage. The existence of G/A polymorphism should be considered a favorable factor promoting a lower aggressiveness of tumor (р<0.05). The existence of G/G major genotype, especially in combination with homozygous mutant allele of MMP1 (1607 1G/2G) gene is an unfavorable factor that is most common in patients with the newly diagnosed colorectal cancer at III-IV stage (р<0.05).

The presence of homozygous mutant G/G genotype of VEGF (C654G) gene may correlate with rapid tumor progression and active metastatic process.

Extension of knowledge of the nature of cancer, peculiarities of its treatment in a particular patient in future will help improve the results of patients’ treatment and to transition of personalized therapy.

About the authors

Evgeniy P. Kulikov

Ryazan State Medical University

Email: moroka1695@yandex.ru
ORCID iD: 0000-0003-4926-6646
ResearcherId: S-1851-2016

Russian Federation, Ryazan

MD, PhD, Professor, Head of the Department of Oncology

Aleksey I. Sudakov

Ryazan State Medical University

Email: moroka1695@yandex.ru
ORCID iD: 0000-0002-6791-9797
SPIN-code: 9307-0078

Russian Federation, Ryazan

Assistant of the Department of Oncology

Aleksandr A. Nikiforov

Ryazan State Medical University

Email: moroka1695@yandex.ru
ORCID iD: 0000-0002-7364-7687
SPIN-code: 8366-5282

Russian Federation, Ryazan

MD, PhD, Associate Professor of the Department of Pharmacology with the Course of Pharmacy of the Faculty of Additional Postgraduate Education

Sergey A. Mertsalov

Ryazan State Medical University

Email: moroka1695@yandex.ru
ORCID iD: 0000-0002-8804-3034

Russian Federation, Ryazan

MD, PhD, Assistant of the Department of Oncology

 

Vladimir A. Grigorenko

Ryazan State Medical University

Author for correspondence.
Email: moroka1695@yandex.ru
ORCID iD: 0000-0002-4664-5723
SPIN-code: 7174-5611
ResearcherId: F-8605-2019

Russian Federation, Ryazan

Clinical Resident of the Department of Oncology

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Copyright (c) 2020 Kulikov E.P., Sudakov A.I., Nikiforov A.A., Mertsalov S.A., Grigorenko V.A.

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