Keywords
interleukin-10 - single-nucleotide polymorphism - breast cancer - RT-PCR - proliferative
activity
Introduction
Breast cancer is the most frequently diagnosed cancer among women and is the second
leading cause of cancer mortality in females. In 2019, breast cancer accounted for
30% of estimated new cancer cases in the United States.[1]
Today, 5 to 10% of all breast cancers are caused by known germline mutations. These
genes are divided into high-risk and low- to moderate-risk susceptibility groups.
Genes like BRCA1, BRCA2, PTEN, TP53, LKB1/STK11, and CDH1 are among high-risk susceptibility genes, while CHEK2, TGFβ1, CASP8, and ATM are among low- to moderate-risk group. Currently, ongoing case–control studies are
dedicated to finding more of such genes.[2] Similarly, this study aims to investigate the role of 1082 single-nucleotide polymorphism
(SNP) in the promoter region of interleukin-10 (IL-10) which is known to influence
its expression.
Even though the human immune system has advanced ways of averting cancer development,
neoplastic cells employ multiple strategies to evade immune detection. One such strategy
is producing immunosuppressive factors that dampen the function of T cells, dendritic
cells, and natural killer cells. Nitric oxide (NO), IL-6, IL-10, tumor growth factor-beta,
indoleamine 2,3-dioxygenase, arginase-1, prostaglandin E2, vascular endothelial growth
factor, and cyclooxygenase-2 are all examples of such factors that help cancers evade
immunity. Among these factors is IL-10 which can impede the function of CD4+ and CD8+
T cells and promote immunosuppressive cells like Treg[3]; hence, promoting cancer development and progression. As an example, it was shown
that in the case of infection with high-risk human papillomavirus strains, the immunosuppressive
properties of IL-10 played a role in facilitating the evasion of immune response by
the pathogen, leading to more progressive cervical disease.[4]
[5]
However, the role of IL-10 in cancer is not so straightforward. IL-10 expression has
also been linked to the downregulation of major histocompatibility complex class I
expression on the cell surface. This change enhances the natural killer cells' detection
of tumor targets, leading to the lysis of tumor cells.[6] Also, a study on the effect of IL-10 on melanoma showed that IL-10 can inhibit macrophage-derived
angiogenic factors and probably act as an antimetastatic agent.[7]
Due to its conflicting immunologic effects, the role of IL-10 in cancer remains uncertain.
IL-10 gene is located on chromosome 1 (1q31-1q32), and SNPs in its promoter region,
-1082 A/G, can alter its expression and therefore can lead to alterations in cancer
susceptibility and cancer progression.[8]
[9]
In this case–control study, we aim to investigate the involvement of this SNP in breast
cancer pathogeneses and its clinical manifestations in the Georgian women population.
Materials and Methods
The study is approved by the Ethics Committee of Tbilisi State Medical University,
Tbilisi, Georgia. Signed informed consent was collected from every study participant.
The study design is a case–control study. Breast cancer patients who were consecutively
admitted to the surgery department in two different oncology hospitals (The First
University Clinic and Cancer Research Center) in Tbilisi, Georgia, were recruited
from 2017 to 2019. Age-matched women who were regularly involved in breast cancer
screening in an outpatient clinic in Tbilisi, and were healthy, were asked to volunteer
as control group members. Clinical information of patients was collected from medical
notes.
The eligibility criteria for the cases were:
-
Biopsy-confirmed diagnosis of breast carcinoma, with no previous history of breast
surgery, and no preoperative chemotherapy or radiation therapy.
-
The age range of 30 to 80 years.
-
Ability to understand the purpose of the study and provide informed consent.
-
Female sex.
-
Being ethnically Georgian.
Eligibility criteria for controls were as followed: No history of previous cancer
diagnosis, no family history of cancer, and minor illnesses were acceptable (e.g.,
common cold, headache). The rest of the criteria (2–5) are identical to that of the
cases.
Due to incomplete data, inappropriate material, or nonmatching diagnosis, 11 patients
were later excluded from the study. In total, 64 patients were involved in the study
along with 64 healthy controls.
Sample Collection and Storage
Blood samples were collected in a vacutainer tube containing ethylenediaminetetraacetic
acid. Genomic deoxyribonucleic acid (DNA) was extracted from the whole blood using
DNA purification kits (Qiagen, United States). DNA concentration was measured using
the fluorometer-based method (Qubit, Thermo Scientific, United States).
Genotyping
SNP genotyping was performed using the TaqMan assay (Thermo Scientific). Each TaqMan
SNP genotyping assay contained sequence-specific forward and reverse primers to amplify
the polymorphic sequence of interest and two TaqMan minor groove binder probes with
nonfluorescent quenchers: one VIC-labeled probe to detect Allele 1(A) sequence and
one FAM-labeled probe to detect Allele 2(G) sequence.
Real-time polymerase chain reaction (PCR) was performed based on standard protocols.
The final volume of PCR reaction was 25 µL, which contained DNA polymerase, forward
and reverse primers with a final concentration of 200 nM, probes with a final concentration
of 250 nM, and 50 ng of genomic DNA. PCR conditions for amplification included polymerase
activation at 95°C for 10 minutes (hold), denaturation at 95°C for 15 seconds, and
annealing/extension at 60°C for 1 minute (cycle 40). The real-time PCR instrument
software plot the results of the allelic discrimination (AD) data as a plot of Allele
1 (VIC dye) versus Allele 2 (FAM dye). The AD plot represents each sample well as
an individual point on the plot. A typical AD plot shows homozygote clusters, a heterozygote
cluster, and no-template controls. The points in each cluster are grouped closely
together and each cluster is located well away from the other clusters.
Statistics
The study and control groups were analyzed separately. All statistical analyses were
performed by GraphPad Prism 9.3.1 for macOS (GraphPad Software, San Diego, California,
United States).
Statistical significance for differences in genotype frequencies was determined by
chi-square and Fisher's exact test, and the level of significance was put at p < 0.05.
An unpaired t-test was used to compare the difference in mean proliferative activity of patients
with AA genotype and patients having the G allele (AG and GG).
To evaluate associations between the SNPs and the risk of cancer, odds ratios (ORs)
and 95% confidence intervals (CIs) were calculated using unconditional logistic regression
analysis. All statistical tests are planned to be two-sided.
Results
Genotyping of IL-10 SNP -1082 A/G was compared between 64 breast cancer cases with
64 healthy controls ([Table 1]). In the case group, the frequencies of AA, AG, and GG genotypes were 29.68, 59.37,
and 10.93%, and in healthy controls 35.93, 54.68, and 9.37%, respectively. The difference
in frequency of genotypes was not statistically significant between the two study
groups ([Table 1], chi-square = 0.5812, p = 0.7478). Neither IL-10 1082 AA (OR = 0.708, 95% CI = 0.213–2.47) nor IL-10 1082
AG (OR = 0.930, 95% CI = 0.266–2.883) were associated with breast cancer. Overall,
there was no association between breast cancer risk and the IL-10 -1082 A/G genotype
([Fig. 1]).
Table 1
Distribution of IL-10 -1082 A/G genotype frequency in breast cancer patients and controls
|
Cases, N (%)
|
Controls, N (%)
|
OR (95% CI)
|
Chi-square
|
p-Value
|
|
AA
|
19 (29.688)
|
23 (35.938)
|
0.708 (0.213–2.47)
|
|
|
|
AG
|
38 (59.375)
|
35 (54.688)
|
0.930 (0.266–2.883)
|
|
|
|
GG
|
7 (10.938)
|
6 (9.375)
|
1.00 Ref
|
0.5812
|
0.7478
|
|
A allele
|
76 (59.375)
|
81 (63.281)
|
0.848 (0.514–1.390)
|
|
|
|
G allele
|
52 (40.625)
|
47 (36.718)
|
1.00 Ref
|
0.4102
|
0.5219
|
Abbreviations: CI, confidence interval; IL-10, interleukin; OR, odds ratio.
Note: The odds ratio of AG/GG to AA is 1.329.
Fig. 1 Frequency of each genotype in cases and controls.
The observed genotype frequencies were in accordance with the Hardy–Weinberg equilibrium.
Among 64 genotyped breast cancer patients, we received pathology reports of 45 patients.
The proliferative activity, estrogen receptor status, progesterone receptor status,
and Her2/neu status were reported and their frequency is shown in [Table 2].
Table 2
Breast cancer status of patients
|
Category
|
Frequency (%)
|
|
Proliferative activity ⩾ 30
|
31.11
|
|
Proliferative activity < 30
|
68.88
|
|
Her2+
|
17.77
|
|
ER+
|
71.11
|
|
PR+
|
68.88
|
Patients' proliferative activity was compared with an unpaired t-test between two groups of AA and AA/AG. Mean proliferative activity in patients
with AA genotype is 20.77 and in AG/GG is 19.34, and there is no statistical significance
between them (t = 0.2575, p = 0.7980). Although when data are put into a box and whiskers plot, there are obvious
outliers in proliferative activity of patients with AG/GG genotype ([Fig. 2]).
Fig. 2 Box and whiskers plot comparing proliferative activity in patients with AA and AG/GG
genotype.
Discussion
The IL-10 SNP at -1082 has been associated with cytokine inducibility and expression
variation. A nuclear protein physically interacts, in an allele-specific manner, with
the promoter region of the IL-10 gene. This protein is poly (ADP-ribose) polymerase
1 (PARP-1). It has been shown that PARP-1 acts as a transcription repressor, with
preferential binding for the A-allele than the G-allele. This difference results in
less expression in the A allele of this gene.[10]
This SNP has been studied in different populations and malignancies, and the results
have been conflicting. A study investigated the result of 22 studies in 13 different
malignancies regarding the SNPs of the IL-10 gene and found a positive association
between IL-10 genotype and disease susceptibility or progression. In some cancers
like cutaneous malignant melanoma and prostate cancer, the genotypes that were associated
with less expression of IL-10 were a risk factor. However, in other cancers like cervical
cancer and hepatocellular cancer, higher expression of IL-10 was a risk factor. The
results are especially more conflicting in breast cancer.[11]
One study on the Indian population[12] and one on the Italian population[13] found that the low IL-10 expressing AA genotype is associated with higher breast
cancer risk.
In this study, the difference we found in the frequency of genotypes between our cases
and controls, who were all Georgian women, was not statistically significant. Our
findings are in accordance with the finding of two studies on Chinese[14] and Iranian[15] populations.
While these differences might be explained by the dual function of IL-10, as an immunosuppressant
(cancer-promoting) and as an antiangiogenic (cancer-inhibiting) agent, it also might
be a result of differences in ethnicity.
This study also compared the proliferative activity between cases harboring the G
allele with those only having the A allele in IL-10 -1082 SNP for the first time.
The proliferative activity was measured by Ki-67 which is a monoclonal antibody that
recognizes proliferating cells.[16] And even though there was no statistically significant difference between the mean
proliferative activity of these two groups, there are obvious outliers in the group
having the G allele with higher proliferative activity shown in [Fig. 1]. It is important to note that a higher Ki-67 index is associated with a poorer prognosis
and higher recurrence rate in breast cancer patients.[17]
Conclusion
This study showed no real association between -1082 A/G SNP and breast cancer risk
in Georgian women. Much larger studies including different ethnicities and studies
combining genotype and gene expression are required to improve our understanding of
the role of IL-10 in cancer development, as this current study comes with limitations
in size and a lack of full phenotype status (for example, the presence of metastasis).
Further understanding of IL-10's role in cancer development can aid us in new cytokine
immunotherapies in malignancies.
