Abstract
Background
Breast cancer is among the leading cause of cancer‐related‐deaths in women, supporting the need for the identification of novel prognostic and predictive biomarkers. Recent studies have identified common genetic variants in a region on chromosome 9p21 associated with an increased risk of developing different cancers. Here, we explored the association of a genetic variant in CDKN2A/B, rs10811661, for the first time in 564 subjects with/without breast cancer.
Method
Genotyping was performed using TaqMan real time PCR method. The associations of this genetic variant with breast cancer risk and pathological information of patients were assessed.
Results
We observed that patients with breast cancer had a higher frequency of TT genotype (P<.001) than control group, which was associated with advanced TNM classification (P=.04) and larger tumor size (P=.014), as detected by the recessive genetic inheritance model. Moreover, the logistic regression under recessive genetic model revealed that breast cancer patients with TT had higher risk of breast cancer, compared to CC/CT genotypes (eg, OR=4.9, 95% CI:1.9‐12, P=.001), after adjusted for potential confounders, age, BMI, and family history.
Conclusion
We demonstrated that patients carrying the TT genotype for CDKN2A/B rs10811661 polymorphism had the increased risk of breast cancer susceptibility. However, further investigations are warranted in a larger and prospective setting to explore the value of this marker as a risk stratification marker in breast cancer.
Keywords: breast cancer, CDKN2A/B, polymorphism, risk marker
1. Introduction
Breast cancer is the second leading cause of cancer‐related deaths in women with poor prognosis.1 Since breast cancer is such a dismal disease, any biomarker that can help to better stratify patients might have a substantial impact on the clinical management of patients. This cancer is characterized by progressive multistep processes, interactions of both genetic and environment factors. Several genes have been reported to be associated with poor outcome in patients with breast cancer (eg, Breast cancer susceptibility genes 1 (BRCA1), BRCA2, HER2, P53 and Ki67). About 5%‐10% of breast cancer cases are supposed to be hereditary 1 and individuals with these mutations have significantly higher susceptibility to developing breast cancer.
Recent genome‐wide association (GWA) studies have reported that genetics variants in a region located on chromosome 9p21.3 are associated with multiple cancers 2, 3, 4, 5 such as ovarian cancer, acute lymphoblastic leukemia, glioma, malignant melanoma, breast cancer, pancreatic cancer, nasopharyngeal neoplasm, and glaucoma.5, 6 The chromosome 9p21.3 region adjacent to the loci encoding the cyclin‐dependent kinase inhibitors CDKN2A (ENSG00000147889) and CDKN2B (ENSG00000147883) is an important susceptibility locus for various diseases with a complex genetic background. Furthermore, this region is also reported to be linked with several other diseases such as cardiovascular diseases,6 type‐2 diabetes,7 periodontitis,8 Alzheimer's disease,9 frailty in the elderly,10 and glaucoma.5 CDKN2A and CDKN2B encode p16 INK4a and p15 INK4b, which inhibit cyclin‐dependent kinase 4 (CDK4) and cyclin‐dependent kinase 5 (CDK5), respectively. CDK4 and CDK5 play an important role in different cell function and regeneration.6 Several genetic polymorphisms have been suggested in the upstream of CDKN2A/B, which might influence the expression of these genes and thereby cell cycle.11 Therefore, the aim of this study was to examine the association of CDKN2A/B rs10811661 polymorphism with breast cancer.
2. Materials and Methods
2.1. Patient samples
In this study, 564 age‐matched Iranian women subjects (92 breast cancer patients and 472 healthy controls) were recruited from Omid or Ghaem hospitals of Mashhad University of Medical Sciences. The patients were histologically confirmed breast cancer between 2013 and 2014. Informed consent was obtained from all participants using protocols approved by the Ethics Committee of the Mashhad University of Medical Sciences.
2.2. DNA extraction and genotyping
Genomic DNAs were extracted from peripheral blood leukocytes using QIAamp® DNA Mini‐Kit (Qiagen, San Diego, CA, USA) according to the manufacturer's protocol. The concentration and purity of DNA samples were determined using the NanoDrop®‐1000‐Detector (NanoDrop‐Technologies, Wilmington, DE, USA). Genotype analysis of CDKN2A/B‐rs10811661 polymorphism was carried out using Taqman®‐probes‐based assay; PCR reactions were carried out in 12.5 μL total volume, using 20 ng of DNA in TaqMan® Universal Master Mix with specific primers and probes (C‐901792‐10 and C‐790057‐10; Applied Biosystems, Foster City, CA, USA). The ABIPRISM‐7500 instrument equipped with the SDS version‐2.0 software was utilized to determine the allelic content of the samples.12, 13
2.3. Statistics
Demographic and clinical information were compared across mutations using Pearson's χ2 tests. The significant prognostic variables in univariate analysis were included in multivariate analyses using Cox's proportional hazards model. This analysis included a step down procedure according to the likelihood ratio test, where Hazard Ratio (HR) was assessed to evaluate the magnitude and the direction of the effect. Appropriate adjustment for false‐positive report probability was carried out according to the Wacholder method. The presence of normal distribution within the subgroups was assessed by Kolmogorov‐Smirnov tests. Continuous variables were evaluated using Student's t tests. The observed genotype frequencies of the CDKN2A/B gen rs10811661 polymorphism were assessed with χ2 tests. The Hardy‐Weinberg equilibrium assumption was assessed by comparing the genotype frequencies by using the Pearson χ distribution. The associations between risk of breast cancer for the CC and CT genotypes, relative to the risk genotype TT homozygote under recessive genetic model were assessed by logistic regression, adjusting for the potential confounders; age, body mass index, family history. Data were analyzed using SPSS‐20 software (SPSS Inc., Chicago, IL, USA). All the analyses were two‐sided and statistical significance was set at P<.05.
3. Results
3.1. Association of the genetic variant with clinical characteristics of population
In order to explore whether there was an association between CDKN2A/2B Rs10811661 (C/T) polymorphism and breast cancer, genotyping was performed in all the subjects using DNA extracted from peripheral blood samples. Genotyping was successfully performed in the all DNA samples and no discrepancies were found in the samples analyzed in duplicate. The distribution of the polymorphism was in Hardy‐Weinberg equilibrium (HWE; P>.05). We then characterized our population based on the genetic information of patients for age, body mass index, SA, tumor size, nodal status, distant metastasis, TNM stage, HER‐2/neu status, Ki62, CEA, and CA153 (Table 1). According to the recessive genetic inheritance model, we found that the TT genotype of was associated with advanced TNM classification (P=.04) and larger tumor size (P=.014), while no association was detected for this genetic variant with distant metastasis, node status, and other clinical features of patients (P>.05).
Table 1.
Genotype distribution of CDKN2A/B rs10811661 polymorphism with respect to clinicopathological features of breast cancer patients under recessive genetic model
| Genotype | ||
|---|---|---|
| CC+CT | TT | |
| Age | 48±17 | 47±11 |
| BMI | 27.8±6 | 28.2±7 |
| Clinical measures | ||
| SA% | ||
| 1+2 | 7.9 | 92.1 |
| 3 | 0 | 100 |
| M | ||
| 0 | 8.1 | 91.9 |
| 1 | 0 | 100 |
| N, % | ||
| 0‐1 | 5.1 | 94.9 |
| 2‐3 | 12 | 88 |
| T*% | ||
| 1‐3 | 5.2 | 94.8 |
| 4 | 33.3 | 66.7 |
| Ki62% | ||
| 0 | 60 | 78 |
| 1 | 40 | 22 |
| Stage**% | ||
| 0 | 40 | 60 |
| 1 | 0 | 100 |
| 2 | 10.3 | 89.7 |
| 3 | 0 | 100 |
| 4 | 7.8 | 92.2 |
| Her2% | ||
| 1+2 | 6 | 94 |
| 3 | 14.3 | 85.7 |
*P=.014, **P=.04.
3.2. Association of the genetic variant with poor prognosis of patients
The distribution of different genotypes (CC, CT, and TT) of CDKN2A/B rs10811661 polymorphism in breast cancer patients and healthy subjects is presented in Table 2. The risk T allele frequency was 85%, and the frequencies of CC, CT, and TT genotypes were 0.03, 22.9, and 74%, respectively, in total population. Patients with breast cancer had a significantly higher frequency of TT genotype (P<.001) than control group (Table 2). Moreover, the logistic regression analysis under recessive genetic model showed that TT carriers had a higher risk of breast cancer (OR=4.9, 95%CI: 1.9‐12, P=.001), with respect to CC/CT genotypes, after adjusted for potential confounders, age, BMI, and family history. However, no statistically significant association was detected for the association of this marker in other genetic models (eg, additive and dominant models).
Table 2.
Association between the genetic polymorphism in CDKN2A/B rs10811661 with breast cancer
| Variant | Genotype distribution | Adjusted model* | |||||||
|---|---|---|---|---|---|---|---|---|---|
| Risk allele | Gene | Case n (%) | Control n (%) | Additive model OR (95%CI) | Recessive model OR (95%CI) | Dominant model OR (95%CI) | HWE P value | ||
| rs10811661 | T | CDKN2B | CC | 2 (2.2) | 16 (3.4) | 2.9 (0.4‐22) | 4.9 (1.9‐12) | 2.27 (0.3‐17) | .2 |
| CT | 7 (7.6) | 122 (25.8) | P add.=.3 | P res.=.001 | P do.=.4 | ||||
| TT | 83 (90.2) | 334 (70.8) | |||||||
4. Discussion
To the best of our knowledge, this is the first study showing the prognostic value of a genetic variant in CDKN2A/B with poor prognosis of patients with breast cancer. Our data demonstrated that patients with TT genotype had a significantly higher risk of breast cancer susceptibility. Moreover, this genotype was related with advanced TNM classification and larger tumor size.
There is growing body of evidence showing the role of CDKN2A/B in breast, ovarian, and bladder and melanoma.2, 3, 4, 5 This link can be explained at least in part by its function in the regulation of cell cycle and apoptosis. It has been shown that the inactivation of CDKN2A by methylation or ANRIL can suppress the activity of tumor suppressor genes encoded by these genes in breast cancer.12 ANRIL indirectly regulates cell proliferation and senescence via three methylations of lysine 27 in chromosome 9P21 (3meH3K27), upfront of CDKN2A/B.13 ANRIL is transcribed as a 3.8 kb lncRNA in the opposite direction from the p15/CDKN2B‐p16/CDKN2A‐p14/ARF gene cluster and has been reported to have a direct role in recruiting PRC2 and PRC1 complexes to specific loci.14 EZH2 (PRC2) and CBX7 (PRC1) are transcriptional repressors of several genes and are specially involved in silencing of the CDKN2B‐CDKN2A‐ARF locus via ANRIL.15 Several genome‐wide association studies have identified the ANRIL gene as a shared genetic susceptibility locus in different cancers.14 However, the potential mechanisms underlying the role of ANRIL in breast carcinogenesis still remained to be elucidated.16 Dębniak et al.,3 suggested the value of CDKN2A as a breast cancer susceptibility gene. They showed that the CDKN2A A148T variant might be contributed to early‐onset breast cancer in Poland.17 In line with these studies, Antoniou and colleagues found the association of the rs1011970, near CDKN2A/CDKN2B, with increased breast cancer risk.18 Another large‐scale case‐control study conducted by Driver et al.,19 examined several polymorphisms within 13 genes involved in the cell cycle pathway with breast cancer risk. They illustrated a significant relationship between four genetics variants within the region of CDKN2A/2B and breast cancer risk.
In aggregate, our finding revealed for the first time a correlation between the genetic polymorphism, rs10811661, in CDKN2A/B gene and breast cancer. In particular, our data showed that this genetic marker was related with tumor grade and tumor size, although further studies in a larger population are needed to explore it values as a potential biomarker in risk stratification or prediction a chance of developing breast cancer.
ShahidSales S, Mehramiz M, Ghasemi F, et al. A genetic variant in CDKN2A/B gene is associated with the increased risk of breast cancer. J Clin Lab Anal. 2018;32:e22190 10.1002/jcla.22190
Funding Information
This study was support by grant from Mashhad University of Medical Sciences.
Mehraneh Mehramiz, Faezeh Ghasemi, and Seyed Amir Aledavood are equally contributed as first author.
Novelty and impact: This study demonstrated the significant association of the CDKN2A/B, rs10811661 polymorphism with outcome in breast cancer patients. This polymorphism can be assessed with a simple blood test, and offers an innovative tool for optimizing palliative chemotherapy in breast cancers. Prospective trials are warranted to validate these findings, which might be applied to the future clinical practice of individualized treatment in selected breast cancer patients.
Contributor Information
Soodabeh ShahidSales, Email: shahidsaless@mums.ac.ir.
Amir Avan, Email: avana@mums.ac.ir, Email: amir_avan@yahoo.com.
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