Introduction
The term triple-negative breast cancer (TNBC) defines breast tumors that do not express estrogen receptors, progesterone receptor, or epidermal growth factor receptor HER2 on immunohistochemical analysis. TNBC refers to about 15–20 % of all breast cancer cases [1‐5].
Molecular profiling indicated that triple-negative breast cancer represents heterogeneous subgroup of breast cancer. Triple-negative breast cancer shares histological and genetic abnormalities with basal-like subtype of breast cancer, however, this overlap is incomplete. Triple-negative breast cancer do not benefit from hormonal therapies or treatments targeted against HER2 [1‐5]. Many of targeted therapeutic agents show promise in early stage studies, but their clinical performance has yet to be definitively proven.
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Molecular epidemiological studies have provided the evidence that an individual’s susceptibility to precancerous lesions and cancer is modulated by both genetic and environmental factors [6, 7]. Genomic rearrangements (translocations, deletions, and duplications) are extremely frequent in breast cancer cells [8‐11]. These rearrangements are believed to result from an aberrant repair of DNA double-strand breaks (DSBs).
Double-strand DNA breaks are the most dangerous DNA damage. If not repaired leads to down-regulation of transcription and various cancers development [12, 13]. DSB are repaired by two mechanisms: recombination (HR) and non-homologous end joining (NHEJ) [14, 15].
A recent study on the Caucasian population has provided the first epidemiological evidence, supporting the association between DSBs repair gene variants and breast cancer development [16].
Polymorphisms in DNA repair genes may alter the activity of the proteins and thus modulate cancer susceptibility [17].
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RAD51 homolog (RecA homolog, E. coli) (S. cerevisiae) plays an important role in homologous recombination via, direct interaction with XRCC2 (X-ray repair cross-complementing group 2), XRCC3 (X-ray repair cross-complementing group 3), BRCA1 (breast cancer-1), BRCA2 (breast cancer-2), and other DNA repair proteins, to form a complex essential for repair of double-strand breaks and DNA cross-links (especially XRCC2 and XRCC3) and for the maintenance of chromosome stability [18‐20].
RAD51 is involved in homologous recombination and repair of double-strand breaks in DNA and DNA cross-links and for the maintenance of chromosome stability. RAD51 gene is highly polymorphic in nature. In the literature, many reports confirm the significance RAD51 gene G135C polymorphism (c. 98 G>C; rs1801320; Genbank Accession Number NT 010194), regarding the risk of breast carcinoma [21‐24].
XRCC2 Arg188His polymorphism (c. 563 G>A; rs3218536, Genbank Accession Number NT 007914) may limit effect on gene activity, although it can modify the breast cancer risk in female patient with low levels of plasma α-carotene or plasma folate [16, 25].
The C722T substitution is the most thoroughly analyzed polymorphism in the XRCC3 gene (c. 722 C>T; rs861539, Genbank Accession Number NT 026437). Although the functional relevance of XRCC3 Thr241Met variation is unknown, some studies have reported that the 722T/T genotype is associated with increased risk of breast cancer [26‐28].
In the present study, the association between the Arg188His polymorphism of XRCC2 gene and Thr241Met polymorphism of XRCC3 gene and triple-negative breast cancer in the population of Polish women was investigated.
Materials and methods
Patients
In the reported study, paraffin-embedded tumor tissue was collected from 70 women with triple-negative breast carcinoma, treated at the Department of Oncology, Institute of Polish Mother’s Memorial Hospital, Lodz, Poland. The age of the patients ranged from 36 to 68 years (the mean age 46.2 ± 10.12). No distant metastases were found in any of the patients at the time of treatment onset. The median follow-up of patients at the time of analysis was 38 months (the range 2–70 months). The average tumor size was 20 mm (the range 17–32 mm). All the tumors were graded by a method, based on the criteria of Scarff–Bloom–Richardson. The demographic data and the pathologic features of the patients are summarized in Table 1. Samples from age-matched, cancer-free women (n = 70) served as control (the mean age 45.41 ± 18.21). Control samples consisted of DNA extracted from normal breast tissue. Normal breast specimens were obtained from patients who had undergone biopsy for benign lesions. An appropriate ethical approval was obtained from the Ethics Committee of the Institute of Polish Mother’s Memorial Hospital, Lodz, Poland.
Table 1
Pathologic features of triple-negative breast cancer patients
Triple-negative breast cancer | Patients (n = 70) | |
|---|---|---|
n
| (%) | |
Scarff–bloom–Richardson stage | ||
I | 20 | 29 |
II | 45 | 64 |
III | 5 | 7 |
Tumor size grade | ||
T1 | 8 | 11 |
T2 | 40 | 57 |
T3 | 18 | 26 |
T4 | 4 | 6 |
Lymph node status | ||
N0 | 32 | 46 |
N1 | 12 | 17 |
N2 | 14 | 20 |
N3 | 7 | 10 |
N4 | 5 | 7 |
The breast tissue samples (cancerous and non-cancerous) were fixed routinely in formaldehyde, embedded in paraffin, cut into thin slices, and stained with hematoxylin/eosin for pathological examination. DNA for analysis was obtained from an archival pathological paraffin-embedded tumor and non-cancerous breast samples which were deparaffinized in xylene and rehydrated in ethanol and distilled water. In order to ensure that the chosen histological material is representative for cancerous and non-cancerous tissue, every tissue sample qualified for DNA extraction was initially checked by a pathologist. DNA was extracted from material using commercially available QIAmp Kit (Qiagen GmbH, Hilden, Germany) DNA purification kit according to manufacturer’s instruction.
Genotyping
Polymorphism of XRCC2 and XRCC3 gene was determined by PCR–RFLP (polymerase chain reaction–restriction fragment length polymorphism), using the appropriate primers (Table 2).
Table 2
Primer sequences for polymerase chain reaction (PCR) analysis
Primer sequence | PCR fragment length (bp) | |
|---|---|---|
XRCC2
| forward 5′-TGTAGTCACCCATCTCTCTGC-3′ | 290 |
reverse 5′-AGTTGCTGCCATGCCTTACA-3′ | ||
XRCC3
| forward 5′-GCCTGGTGGTCATCGACTC-3′ | 552 |
reverse 5′-ACAGGGCTCTGGAAGGCACTGCTCAGCTCACGCACC-3′ |
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Determination of XRCC2 genotype
The 25 μL PCR mixture contained 100 ng of DNA, 12.5 pmol of each primer, 0.2 mmol/l of dNTPs, 2 mmol/l of MgCl2, and 1 U of Taq DNA polymerase. Thermal cycling conditions were as follows: initial denaturation step at 94 °C, 30 cycles at 94 °C for 30 s, and 30 s at 60 °C annealing temperature, and at 72 °C for 1 min. The final extension was performed at 72 °C for 7 min. The 290 bp amplified product was digested overnight with 1 U of HpnI (New England Biolabs, Ipswich, MA, USA) at 37 °C. The wild-type allele Arg was identified by the presence of single band of 290 bp, while the mutant allele His was represented by 148 and 142 bp bands.
Determination of XRCC3 genotype
XRCC3 gene polymorphism was determined by PCR–RFLP, using codon 241 primers. The 25 μL PCR mixture contained 100 ng of DNA, 12.5 pmol of each primer, 0.2 mmol/l of dNTPs, 2 mmol/l of MgCl2, and 1 U of Taq DNA polymerase. Thermal cycling conditions were the following: 94 °C for 60 s, 56 °C for 30 s, and then 72 °C for 40 s, repeated in 30 cycles. The 552 bp amplified product was digested overnight with 5 U of NlaIII (New England Biolabs, Ipswich, MA, USA) at 37 °C. The wild-type allele Thr was identified by the presence of two 239 and 313 bp bands, while the mutant allele Met was represented by 105, 208, and 239 bp bands.
Statistical analysis
Genotype frequency deviations were assessed for each polymorphism, comparing Hardy–Weinberg equilibrium values with control values by the standard Chi square test. Genotype frequencies in the study cases and the controls were compared by the Chi square test. Genotype specific risks were estimated as odds ratios (ORs) with associated 95 % intervals (CIs) by unconditional logistic regression. p values <0.05 were considered significant. All the statistical analyses were performed, using the STATISTICA 6.0 software (Statsoft, Tulsa, OK, USA).
Results
The genotype frequency of the XRCC2 Arg188His polymorphism in the TNBC patients and controls is summarized in Table 3. It can be seen from the Table that there are significant differences in the frequency of genotypes (p < 0.05) between the two investigated groups. A weak association was observed between triple-negative breast carcinoma occurrence and the presence of at least one 188His allele. A stronger association was observed for 188His/His than for 188Arg/His heterozygous variant. In case of the Arg188His polymorphism of XRCC2 gene, the distribution of the genotypes in the patients differed significantly from one expected from the Hardy–Weinberg equilibrium (p < 0.05).
Table 3
Distribution of 188Arg/Arg, 188Arg/His, and 188His/His genotypes and frequencies of the Arg and His alleles in patients with triple-negative breast cancer and controls
XRCC2 Arg188His | TNBC patients (n = 70) | Controls (n = 70) | OR (95 % CI)a
|
p
b
| ||
|---|---|---|---|---|---|---|
Number | (%) | Number | (%) | |||
188Arg/Arg | 12 | 17 | 18 | 26 | 1.00 Ref | |
188Arg/His | 8 | 12 | 40 | 57 |
0.30 (0.10–0.86)
| 0.042 |
188His/His | 50 | 71 | 12 | 17 |
6.25 (2.38–16.39)
| 0.0003 |
188Arg | 32 | 23 | 76 | 54 | 1.00 Ref | |
188His | 108 | 77 | 64 | 46 |
4.00 (2.39–6.71)
| <.0001 |
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No statistically significant differences were observed in genotype frequencies of XRCC3 Thr241Met polymorphism between the control group and the TNBC patients (see Table 4). Among the patients, all genotype distributions did not differ significantly (p > 0.05) from those expected by the Hardy–Weinberg equilibrium.
Table 4
Distribution of 241Thr/Thr, 241Thr/Met, and 241Met/Met genotypes and frequencies of the Thr and Met alleles in patients with triple-negative breast cancer and controls
XRCC3 Thr241Met | TNBC patients (n = 70) | Controls (n = 70) | OR (95 % CI)a
|
p
b
| ||
|---|---|---|---|---|---|---|
Number | (%) | Number | (%) | |||
241Thr/Thr | 19 | 27 | 15 | 21 | 1.00 Ref | |
241Thr/Met | 35 | 49 | 35 | 50 | 0.78 (0.34–1.79) | 0.718 |
241Met/Met | 16 | 23 | 20 | 29 | 0.63 (0.24–1.62) | 0.475 |
241Thr | 73 | 52 | 65 | 46 | 1.00 Ref | |
241Met | 67 | 48 | 75 | 54 | 0.79 (0.49–1.27) | 0.402 |
Histological grading was related to XRCC2 Arg188His and the XRCC3 Thr241Met polymorphisms. Histological stages were evaluated in all the cases (n = 70). There were as follows: stage I—20 cases, stage II—45 cases, and stage III—5 cases. Stages II and III were accounted together for statistical analysis (see Table 5). Some correlation was observed between the XRCC2-Arg188His and XRCC3-Thr241Met polymorphisms and triple-negative breast cancer invasiveness. An increase was observed, regarding 188Arg/His heterozygotes frequency (OR 2.45; 95 % CI 0.66–9.02, p = 0.289) and 241Thr/Met heterozygotes (OR 2.50; 95 % CI 0.68–9.11, p = 0.267) in stage I patients, according to Scarff–Bloom–Richardson classification. That increase was, however, not statistically significant.
Table 5
Dependence of genotypes and frequencies of XRCC2 and XRCC3 gene polymorphism alleles on tumor stage in triple-negative breast cancer patientsa
Stageb
| Triple-negative breast cancer patients | |||
|---|---|---|---|---|
I (n = 20) | II + III (n = 50) | OR (95 % CI)c
|
p
d
| |
XRCC2 Arg188His | ||||
188Arg/Arg | 4 (20) | 14 (28) | 1.00 Ref | |
188Arg/His | 14 (70) | 20 (40) | 2.45 (0.66–9.02) | 0.289 |
188His/His | 2 (10) | 16 (32) | 1.09 (0.29–4.08) | 0.588 |
188Arg | 22 (55) | 48 (48) | 1.00 Ref | |
188His | 18 (45) | 52 (52) | 0.76 (0.36–1.57) | 0.571 |
XRCC3 Thr241Met | ||||
241Thr/Thr | 4 (20) | 15 (30) | 1.00 Ref | |
241Thr/Met | 14 (70) | 21 (42) | 2.50 (0.68–9.11) | 0.267 |
241Met/Met | 2 (10) | 14 (28) | 0.53 (0.08–3.39) | 0.417 |
241Thr | 22 (55) | 51 (51) | 1.00 Ref | |
241Met | 18 (45) | 49 (49) | 0.85 (0.40–1.77) | 0.806 |
Table 6 shows the distribution of genotypes and the frequency of alleles in patients with (N+) and without (N−) lymph node metastases. A tendency for a decreased risk of breast cancer was observed with the occurrence of 188His/His genotype and 188His allele of XRCC2 and 241Met/Met genotype and 241Met allele of XRCC3 polymorphism. That decrease was, however, not statistically significant (p > 0.05) (see Table 6). There were no differences either in the distribution of genotypes or the frequency of alleles in the group of patients with different tumor size (Table 6).
Table 6
XRCC2 and XRCC3 gene polymorphism and triple-negative breast cancer progressiona
TNBC patients (n = 70) | TNBC patients (n = 70) | |||||
|---|---|---|---|---|---|---|
Tumor size | Node status | |||||
T3 + T4
N = 22 | T1 + T2
N = 48 | OR (95 % CI)a
| N+ (n = 38) | N− (n = 32) | OR (95 % CI)b
| |
XRCC2 Arg188His | ||||||
188Arg/Arg | 8 (36) | 17 (35) | 1.00 Ref | 12 (32) | 10 (31) | 1.00 Ref |
188Arg/His | 8 (36) | 18 (38) | 2.51 (0.57–11.1) | 16 (42) | 11 (34) | 1.21 (0.38–3.78) |
188His/His | 6 (28) | 13 (27) | 2.61 (0.54–12.3) | 10 (26) | 11 (34) | 0.75 (0.22–2.51) |
188Arg | 24 (55) | 52 (54) | 1.00 Ref | 40 (53) | 31 (48) | 1.00 Ref |
188His | 20 (45) | 44 (46) | 0.98 (0.48–2.01) | 36 (47) | 33 (52) | 0.84 (0.43–1.64) |
XRCC3 Thr241Met | ||||||
241Thr/Thr | 7 (32) | 17 (35) | 1.00 Ref | 13 (35) | 10 (31) | 1.00 Ref |
241Thr/Met | 9 (41) | 19 (40) | 1.15 (0.35–3.76) | 15 (39) | 10 (31) | 1.15 (0.36–3.64) |
241Met/Met | 6 (27) | 12 (25) | 1.21 (0.32–4.53) | 10 (26) | 12 (38) | 0.64 (0.19–2.07) |
241Thr | 23 (52) | 53 (55) | 1.00 Ref | 41 (54) | 30 (47) | 1.00 Ref |
241Met | 21 (48) | 43 (45) | 1.12 (0.55–2.30) | 35 (46) | 34 (53) | 0.75 (0.38–1.46) |
Discussion
According to our data, it is the first time that polymorphisms in XRCC2 and XRCC3 genes involved in the DNA repair pathway were analyzed in the population of Polish women with TNBC. The combined effect of XRCC2 and XRCC3 polymorphisms on TNBC occurrence was not investigated before. The study was performed on an ethnically homogenous population, which may improve our knowledge, regarding to what an extent the genotype–phenotype relationship variations are population-related.
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The polymorphisms, chosen for the study, had previously been shown to have functional significance and to be responsible factors for low DNA repair capacity phenotype, characteristic for patients with cancer including those with breast carcinoma [20].
The genes involved in DNA repair and in the maintenance of genome integrity play a crucial role in providing protection against mutations that may lead to cancer [29].
XRCC2 and XRCC3 proteins are structurally and functionally related to RAD51, which plays an important role in the homologous recombination, the process being frequently involved in cancer transformation [30].
RAD51, XRCC2, and XRCC3 gene are highly polymorphic. A single nucleotide polymorphism, 135G/C, has been identified in the 5′ untranslated region of the RAD51 gene and has been shown to influence gene transcription activity [31]. As it was mentioned in the Introduction above, the reports on the relationship between RAD51 G135C polymorphism and breast cancer incidence are suggest that the RAD51 135C variant allele was associated with an increased risk of female breast cancer [22, 23, 32, 33].
By contrast, Brooks et al. [34] showed that RAD51 gene variants were found to be not associated with breast cancer risk.
Other studies have shown that the RAD51 135C variant allele was associated with an increased risk of female breast cancer [35‐37].
135C/C genotype may be associated with an elevated tumor risk among the European populations, regarding sporadic breast cancer [36]. Similar results were obtained in the Polish population [38].
In our earlier study, RAD51 135C allele variant was associated with an elevated risk of triple-negative breast cancer in the Polish women [39].
It is possible that the presence of C allele remains in a linkage disequilibrium with another, so far unknown, mutation located outside the coding region in the RAD51 gene, which may be important, regarding RAD51 concentrations in plasma.
In the presented study, XRCC2 Arg188His genotype was associated with an elevated risk of triple-negative breast cancer in the Polish population. There was a 6.25-fold increased risk of TNBC for the individuals, carrying XRCC2-188His/His genotype, compared with subjects carrying XRCC2-188Arg/Arg, 188Arg/His genotype, respectively. XRCC2 Arg188His polymorphism was not related, either to tumor size or cancer type or grade.
In the reported study, the Arg188His polymorphism of XRCC2 gene and Thr241Met of XRCC3 were correlated with breast carcinoma progression. Arg188His and Thr241Met heterozygote were associated with an increased risk of stage I breast cancer.
However, other literature data were also found [40‐42]. No significant associations were observed between the Thr241Met and breast cancer in Iowa and Cypriot women (40, 43).
In the Polish population, Thr241Met genotype of XRCC3 polymorphism increased the risk of breast cancer development [41, 42, 44].
Similar to our observation, the recent reports demonstrate that XRCC3 Thr241Met allele seems associated with an elevated breast cancer risk in non-Chinese subjects (28).
The role of position 188 in the aminoacid chain for XRCC2 protein functionality is still unknown. The several data suggest that XRCC2 Arg188His polymorphism is not directly associated with breast cancer risk [45, 46].
In conclusion, the reported study is another evidence for the significance of Thr241Met and Arg188His genotype in breast carcinoma staging.
The obtained data show that Arg188His and Thr241Met polymorphisms of XRCC2/3 genes may be associated with the risk of triple-negative breast carcinoma occurrence. On the other hand, a protective effect was observed of all the polymorphisms in the patients without (N-) lymph node metastasis. The obtained data suggest that the reported study may be the first observation of the polymorphisms in XRCC2 and XRCC3 genes, involved in the DNA repair pathway, to be associated with triple-negative breast carcinoma risk in the population of Polish women.
Finally, it is postulated that these polymorphisms may be used as predictive factors for TNBC in the Polish female population. Further studies, conducted on a larger group, are suggested to clarify this point.
Conflict of interest
The authors declare no conflict of interest.
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