Substitutions of the NBS1 gene and clinicopathological characteristics of young breast cancer patients

INTRODUCTION

It is estimated that breast cancer (BC) comprises nearly 25% of all cancers in women and it is one of major causes of women cancer mortality worldwide (Ferlay et al., 2012). Preventive breast cancer prophylactic, new diagnostics, and treatment options have improved survival of breast cancer but there is still a need of better understanding of the  disease. Realisation of molecular breast cancer mechanisms established on the basis of genetic differences in patients is important to individual medicine, therapy optimization, and cancer prognosis. Recent studies have focused on genetic factors, which increase cancer risk, but there is little data about the association of the genetic variation with prognostic aspects of the malign disease (Lima et al., 2019).

Nibrin (95 kDa), the expression product of the  Nijmegen Breakage Syndrome 1 (NBS1) gene [OMIM 602667],  –  is responsible for maintaining genome stability. Hypomorphic mutations that lead to decreased functions of the NBS1 gene are responsible for the Nijmegen Breakage Syndrome (NBS), a  rare autosomal recessive syndrome characterized by microcephaly, growth retardation, immunodeficiency, and predisposition to different malignancies. Mostly lymphoid and haematological cancers are observed, however, an increased susceptibility to solid tumours for homozygotes or heterozygotes of the mutated NBS1 was reported (Cybulski et al., 2004; Steffen et al., 2004). Recently published data has provided the  information that the  mutated NBS1 gene has influence on breast cancer risk mainly in Slavic populations (Cybulski et al., 2019; Rusak et al., 2019). The highest rate of heterozygous carriers of present gene mutations is noted in Eastern and Central Europe, especially in Poland, the Czech Republic and Ukraine (Huzarski et al., 2013).

It is estimated that approximately 0.5% of the Eastern European population are heterozygous for 5  bp deletion 657_661 delACAAA (c.657_661del) in NBS1 (Varon  et  al., 2000). This truncating germ line mutation that reside in 6th NBS1 gene exon leads to formation of two alternative forms of nibrin with a lower molecular weight, of approximately 26 and 70  kDa (di Masi, Antoccia, 2008). Therefore majority of studies in this restricted European region analysed the  NBS1 c.657_661del mutation association with breast cancer predisposition. Although an elevated risk of breast cancer has been observed among carriers of c.657_661del, it was not confirmed to be associated with the course of the disease. According to c.657_661del mutation status, Huzarski et al. (2013) identified that mutation carriers and non-carriers presented with similar clinicopathological breast tumour characteristics and survival rate.

Since the identification of the NBS1 gene, many other mutations have been discovered. Recently, missense substitutions p.R215W and p.I171V have been suggested to be breast cancer susceptibility factors (Cybulski et al., 2004; Bogdanova  et  al., 2008a; Roznowki  et  al., 2008). In the study by Roznowski et al. (2008), the  association between clinicopathological tumour features and mutation status was analysed. Although no statistically significant association was reported, it was suggested that p.I171V mutation could be a  negative prognostic factor.

It has been suggested that NBS1 polymorphisms contribute to the  aetiology of malignancies. One of the  most commonly studied NBS1 polymorphism is c.8360G>C (rs1805794), which has been determined to increase the risk of specific cancer in relation to person’s ethnicity (Lu et al., 2009; He et al., 2014). Lu  et  al. (2006) findings suggest that NBS1 c.8360G>C polymorphism and haplotypes may contribute to the aetiology of sporadic breast cancer in young non-Hispanic white woman. According to Sun et al. (2007), CC genotype of this polymorphism is associated with increased neutropenic fever in breast cancer patients. Also, these studies assessed the  contribution of c.30537G>C (rs1805787) polymorphism to increased breast cancer risk, but the  results were inconclusive (Lu  et  al., 2006; Sun et al., 2007).

In the present study, we performed the analysis for NBS1 gene mutations (c.657_661del, p.I171V, p.R215W) and polymorphisms (c.8369G>C, c.30537G>C) for young women with breast cancer diagnosis. The  majority of the  studies analyse the  relative risk of breast cancer in the  case of current gene substitutions (Lu et al., 2006; Steffen et al., 2006; Bogdanova et al., 2008b; di Masi, Antoccia, 2008; Wang  et  al., 2010). However, the  aim of this research was to determine the possible association between NBS1 and clinicopathological features together with BC progression.

MATERIALS AND METHODS

Study population

Biological material was obtained from 80 breast cancer patients. In genetic analysis, young premenopausal women (≤50 years of age) with I or II stage breast cancer were involved. All samples were analysed at the Oncology Research Laboratory of the Oncology Institute of the Lithuanian University of Health Sciences. The  study was approved by the  local Biomedical Research Ethical Committee (protocol number BE-2-13). Patients provided written informed consent for the participation in the study.

The data on tumour clinicopathological characteristics was obtained from medical records.

Tumour oestrogen, progesterone receptors, and HER2 (human epidermal growth factor receptor 2)  status were determined with immunohistochemistry. In the case of HER2, it was considered positive when immunohistochemically detected HER2 (3+) or (2+) amplification was confirmed by positive Fluorescence in situ hybridization. Testing for HER2 status gave as a score of 0 to 3+ that measured the amount of HER2 proteins on the surface of the cells from breast tumour biopsy. Score from 0 to 1+ was considered as HER2-negative. A  score of 2+ and 3+ was considered HER2-positive.

The majority of breast tumours were not larger than 2  cm (63.8%). Most of selected young breast cancer patients (85%) had negative tumour stromal lymphocyte infiltration. Approximately half of the cases (48.8%) were positive for lymph node involvement. The majority of the  tumours (68.8%) were well to moderate differentiated (grade 1 (G1) or grade 2 (G2)). Low grade (G1) is well differentiated breast cancer. Cancer cells look like normal breast cells and grow (spread) slowly. Intermediate grade (G2) is moderately differentiated breast cancer. Cancer cells look slightly different to normal breast cells. High grade (G3) is poorly differentiated breast cancer. Cancer cells look very different to normal breast cells. In the case of tumour receptor expression, almost half patients were positive for estrogen (51.2%) and progesterone (46.3%) receptors, HER2 amplification was determined in 22.5% of tumours. Most of breast cancer patients had Luminal A tumour subtype (47.5%). Luminal B, HER2 and triple negative (tumours that does not have any of the  receptors that are found in breast cancer, e.g. oestrogen, progesterone receptors, and HER2) breast cancer subtypes were determined in 12.5%, 10.0%, and 30.0% of selected cases, respectively. During the follow-up period (of at least two years), 27.5% patients were documented with a local or systemic disease progression.

Genetic analysis

DNA was isolated from peripheral blood leukocytes using commercially available Gene-JET genomic DNA purification kit, according to manufacturer’s recommendations (Thermo Fisher Scientific Baltics, Lithuania). Polymerase chain reaction (PCR) primer sequences and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) conditions for genotyping are listed in Table 1. All restriction enzymes for PCR-RFLP were from Thermo Fisher Scientific Baltics, Lithuania. Genetic testing for current gene mutation or polymorphism was done as described above, with some modifications when necessary (Resnick  et  al., 2003; Lu et al., 2006; Bogdanova et al., 2008a; Bogdanova et al., 2008b).

Table 1. Primer sequences, PCR-RFLP conditions for the analysis of NBS1 mutations and polymorphisms

* WT – wild type.

Statistical analysis

Statistical analysis was done with SPSS 20.0 for Windows (SPSS, Inc., Chicago, IL, USA). In the  case of qualitative variables, comparison was made by the 2-sided Pearson’s Chi-square or 2-sided Monte Carlo exact test (in the cases where >25% of the expected value were <5). In order to calculate the crude and adjusted odds ratio (OR) and their 95% confidence intervals (CIs) (with and without adjustment for cancer diagnosis age), logistic regression analysis was used. Results were statistically significant at P value lower than 0.05.

RESULTS

Among the 80 patients, no carriers of the NBS1 c.657_661del, p.R215W, and p.I171V mutations were found. NBS1 c.8360G>C genotyping identified 11 patients with GG (13.75%), 38 with GC (47.5%), and 31 with CC (38.75%). Regarding c.30537G>C polymorphism, 46 patients were GG (57.5%), 29 GC (36.25%), and five with CC genotype (6.25%). The genotypes of NBS1 c.8360G>C and c.30537G>C were under Hardy-Weinberg Equilibrium. The NBS1 polymorphism genotype distribution between pathological characteristics is summarized in Table 2. Our descriptive data indicate that c.8360G>C and c.30537G>C variants do not statistically contribute to breast cancer characteristics. Dominant and recessive models showed no significant associations with studied characteristics in our study (Table 3).

Table 2. The distribution of tumour clinicopathological characteristics by NBS1 genotype

The  association between NBS1 polymorphisms and clinicopathological characteristics was also assessed by the odds ratio with its corresponding 95% confidence interval. The logistic regression analysis showed no significant differences between NBS1 polymorphism genotype and pathological features when the model was adjusted for the diagnosis age. However, there was a significant association between c.8360G>C polymorphism and clinicopathological characteristics in the  model without adjustment for the age of breast cancer diagnosis (Table 4). It was determined that heterozygous carriers of NBS1 8360 GC genotype had a 2.885-fold decreased risk of low grade (well-differentiated) (OR = 2.885, 95% CI 0.173–0.735, P = 0.005) tumour compared with those with GG genotype. The homozygote CC genotype of this polymorphism carriers had a  2.186-fold (OR  =  2.186, 95% CI 0.188–0.888, P  =  0.024) decreased risk of low grade (G1+2) tumour differentiation. 8360 CC genotype (OR = 3.034, 95% CI 0.156–0.778, P = 0.010) significantly increased the chances of HER2 amplification compared to GG genotype. The data indicated that patients with 8360 GC genotype had a  higher risk for breast cancer progression (OR = 1.673, 95% CI 0.233–0.915, P = 0.027). The carriers of homozygote 8360 CC had approximately six times higher risk for the disease progress (OR = 5.946, 95% CI 0.098–0.585, P = 0.002) when compared to GG genotype. The prevalence of triple negative breast cancer subtype was significantly higher in individuals with NBS1 8360 CC genotype (OR = 2.186, 95% CI 0.188–0.888, P = 0.024). Regarding c.30537G>C, none of the genotypes had any significant influence on pathological characteristics.

Table 3. NBS1 polymorphisms genotypes models association with clinicopathological breast cancer characteristics

* Dominance of the alleles can be assumed by combining the heterozygote and one of the homozygotes as a single group. In c.8360G>C polymorphism (same with c.30537G>C variant), C is the “risk” allele. In the recessive model, two copies of allele C required to affect breast cancer prognosis, i.e., GG+GC vs CC are used in the analysis. The dominant effect of the “risk” C allele is described by comparing GC+CC vs GG.

Table 4. Odds ratio for the associations of NBS1 c.8360G>C polymorphism with clinicopathological features of breast cancer

* Only statistically significant associations are shown.

DISCUSSION

Well-designed analysis of mutations or polymorphisms of the NBS1 gene or other DNA repair genes could potentially identify prognostic and predictive factors for breast cancer. c.657_661del mutation in the NBS1 gene clearly has association with an increased breast cancer risk, missense substitutions, e.g., p.I171V and p.R215W, may represent cancer susceptibility alleles with lower penetrance (Bogdanova et al., 2008a).

Several studies have investigated not only the risk of breast cancer but also clinical characteristics of the  disease in the  case of NBS1 mutation. Previous report about the  influence of NBS1 gene p.I171V mutation on histological and clinical breast cancer features suggested accumulation of the  negative prognostic factors (Roznowski  et  al., 2008). In the  study by Roznowski and colleagues (2008), the carriers of p.I171V showed a trend of a more aggressive cancer phenotype: higher histological grade, HER2 overexpression, lower oestrogen receptor expression, and familial history of malign disease. According to Nowak et al. (2008), NBS1 p.I171V mutation may be a general susceptibility factor to solid tumours. The  study by Huzarski  et  al. (2013) showed that NBS1 c.657_661del did not affect breast cancer prognosis. In this case-control analysis, tumour size, grade, receptors, and lymph node status were also included. No statistically significant differences between mutation carriers and non-carriers were observed. Moreover, several studies have investigated the relation between NBS1 gene c.8360G>C and c.30537G>C polymorphisms and cancer risk, but the results were inconclusive (He  et  al., 2014). Sun  et  al. (2007) revealed that NBS1 c.8360G>C polymorphism CC genotype may be one of the predictive neutropenic fever factors for breast cancer patients. All mentioned clinicopathological breast cancer characteristics and NBS1 gene status association analysis included case-control groups, but there is still a  need of prospective studies with a larger sample size and with broader ethnic diversity.

In the present study, we investigated the possible association between mutations and polymorphisms in the  NBS1 gene and clinicopathological characteristics of breast cancer. Compared with other studies (Roznowski et al., 2007; Huzarski et al., 2013; Rusak et al., 2019), our study is rather unique. We included young (≤50 years of age) premenopausal women with I or II stage breast cancer diagnosis. The stage of breast cancer is a strong prognostic factor; in order to avoid its influence, we chose women with an early stage of breast cancer. To reveal the relation between NBS1 gene substitutions and clinicopathological characteristics, our analysis involved features that represent breast cancer phenotype and disease progress. No carriers of c.657_661del, p.I171V and p.R215W mutations were found. It might be due to the  relatively young breast cancer patients’ age, as it is known that mutations in the  NBS1 gene more often influence malign disease in elderly patients. In the  current study, some statistically significant associations were determined. NBS1 c.8360G>C polymorphism GC and CC genotypes had effect on the histological tumour grade. It was observed that heterozygote GC and homozygote CC genotype holders had an increased risk for a  higher histological grade. Furthermore, we revealed that CC genotype of c.8360G>C increased the odds of HER2 overexpression, which is a strong prognostic breast cancer factor. It was determined that current polymorphism heterozygous GC and homozygous CC genotypes increased the chances of breast cancer progression if compared with GG genotype. According to our results, NBS1 c.8360G>C polymorphism CC genotype increased the odds of triple negative breast cancer. In the  case of c.30537G>C polymorphism, no significant associations with breast cancer characteristics were found.

Unfortunately, there are some difficulties concerning the  conclusion about the  NBS1 gene status and breast cancer clinicopathological characteristics association because of small group of analysed patients compared with studies conducted by other researchers. We have to admit that the second limitation of our study is a short follow-up period of our patients.

CONCLUSIONS

Our data suggest that NBS1 gene c.8360G>C polymorphism may be associated with breast cancer aggressiveness in young breast cancer patients. Further work is warranted to investigate the effect of the NBS1 status on breast cancer prognosis and malignancies risk.

COMPETING INTEREST

The authors declare that they have no competing interests.

ACKNOWLEDGEMENTS

We would like to thank the  patients for their participation in this study.

References

Roberta Vadeikienė, Aistė Savukaitytė, Rasa Ugenskienė, Jurgita Gudaitienė, Elona Juozaitytė

NBS1 GENO POKYČIŲ IR KLINIKINIŲ PATOLOGINIŲ CHARAKTERISTIKŲ RYŠYS KRŪ-TIES VĖŽIU SERGANČIŲ JAUNŲ PACIENČIŲ GRUPĖJE