Genetic predisposition to male breast cancer in Poland

Background Breast cancer in men accounts for fewer than 1 % of all breast cancer cases diagnosed in men and women. Genes which predispose to male breast cancer include BRCA1 and BRCA2. The role of other genes is less clear. In Poland, 20 founder mutations in BRCA1, BRCA2, CHEK2, PALB2, NBN, RECQL are responsible for the majority of hereditary breast cancer cases in women, but the utility this genes panel has not been tested in men. Methods We estimated the prevalence of 20 alleles in six genes (BRCA1, BRCA2, CHEK2, PALB2, NBN, RECQL) in 165 Polish male breast cancer patients. We compared the frequency of selected variants in male breast cancer cases and controls. Results One of the 20 mutations was seen in 22 of 165 cases (13.3%). Only one BRCA1 mutation and two BRCA2 mutations were found. We observed statistically significant associations for PALB2 and CHEK2 truncating mutations. A PALB2 mutation was detected in four cases (OR = 11.66; p < 0.001). A CHEK2 truncating mutation was detected in five cases (OR = 2.93;p = 0.02). Conclusion In conclusion, we recommend that a molecular test for BRCA1, BRCA2, PALB2 and CHEK2 recurrent mutations should be offered to male breast cancer patients in Poland. Supplementary Information The online version contains supplementary material available at 10.1186/s12885-021-08718-3.

In the current study, we evaluated the frequency of 20 recurrent mutations in six genes (BRCA1, BRCA2, CHEK2, PALB2, NBN and RECQL) in 165 men diagnosed with breast cancer in Poland.

Patients
Patients were selected from a registry of breast cancer cases held at the Hereditary Cancer Center in Szczecin. We identified 186 men with breast cancer from a registry of 25,000 breast cancer cases diagnosed in Poland. Patients had a genetic consultation between 2000 and 2019 at the Hereditary Cancer Center in Szczecin. During the patient interview a written, informed consent and a blood sample were obtained from all subjects. All patients were of European ancestry and ethnic Poles.
Because of the quality of the DNA we were able to include 165 men in the study (88.7%). Clinical data including family history (cancer cases in first and seconddegree relatives) and tumor characteristics were obtained  during an interview with the patient and from medical records ( Table 1). All clinical data were obtained in more than 70% of cases.

Controls
The frequencies of BRCA1, CHEK2, PALB2, NBN in cancer-free controls were taken from previous studies [29,30,56]. The frequency of mutations in the BRCA1 and NBN genes were determined in the group 4000 samples described by Lener et al. [56]. The frequency of mutations in the CHEK2 gene were determined in the group of 5496 samples described by Cybulski et al. [29]. The frequency of mutations in the PALB2 gene were determined in the group of 4702 samples described by Cybulski et al. [30].

Statistical analysis
We estimated the odds ratios for selected recurrent mutations. We compared the prevalence of the ten different mutant alleles in BRCA1, CHEK2, PALB2, NBN in the study group and in control groups. Odds ratios (OR) were generated from two-by-two tables and statistical significance was assessed using Fisher exact test or Chisquared test with Yates correction. ORs were used as estimates of relative risk.

Results
There were 165 men with breast cancer included in the study. The median age of diagnosis was 62 years (range 26-89 years). Twenty-five cases were diagnosed before age 50 (15.2%) ( Table 1). The majority of cases were diagnosed with ductal carcinoma (89.6% of cases), followed by lobular (4.2% of cases) and papillary cancer (3.7% of cases). 91.4% were estrogen receptor (ER) positive and 82.7% were progesterone receptor (PR) positive. Triple negative receptor status was found in 8.9% cases. Bilateral breast cancer was diagnosed in two patients (1.2%) ( Table 1).
Multiple primary cancers were diagnosed in 20 of 165 patients (12.1%). Prostate cancer was diagnosed in five cases (3.0%) and colorectal cancer was diagnosed in four patients (2.4%). Approximately one-half of the patients had a positive family history of cancer: 31.5% for breast and/or ovarian cancer and 36.4% for other cancers. The clinical features of breast cancer diagnosed in carriers of mutations and non-carriers are presented in Table 1.
We did not find statistically significant differences between mutation carriers and non-carriers (p > 0.5), except for a higher incidence of bilateral breast cancer in PALB2 mutation carriers (p = 0.004).
One of twenty founder mutations was diagnosed in 22 of 165 men (13.3%) ( Table 2). A CHEK2 mutation was found in 14 patients (8.5%). The most commonly detected CHEK2 allele was the missense c.470C > T mutation, which was found in nine subjects (5.5%). A protein truncating mutation in CHEK2 gene was found in five patients (3.0%). A PALB2 mutation was seen in four patients (2.4%). A BRCA2 mutation was found in two patients (1.2%) and a BRCA1 mutation was found in only one patient (0.6%). One mutation was found in NBN (0.6%) and no mutations were found in RECQL.
A mutation was found in 20% of men diagnosed before age 50 and in 12.9% of men diagnosed after age 50. A mutation was found in 9.7% of men with ductal carcinoma and 15.2% of men with other histopathological subtypes. Mutations were more common in familial cases (17.3%) than in non-familial cases (11.5%).
Compared to the frequency of mutations in cancerfree controls from our previous studies, we observed statistically significant associations for PALB2 (OR = 11.66 p < 0.001) and for CHEK2 truncating mutations (OR = 2.93; p = 0.02) ( Table 3).
In two men, three primary cancers were diagnosed (1.3%). Both of these patients were diagnosed with bilateral breast cancer and both carried a mutation in the PALB2 gene. a -for BRCA1, CHEK2, PALB2, NBN mutation frequencies in cancer-free controls were from our previous studies [29,30,56].

Discussion
A founder mutation of BRCA1, BRCA2, CHEK2, PALB2 and NBN was found in 13.3% of men with breast cancer in Poland. The mutation frequency was similar in familial and non-familial cases and in young men and older men.
This study supports the recommendation that a panel test be offered to all Polish men with breast cancer. In a previous study we have shown that 20 founder mutations account for 85% of all mutations in Polish women with hereditary breast cancer and were detected in 46% cases [27].
Our study is the first that evaluated the frequency of mutations in the PALB2 gene in men with breast cancer  from Poland, which is populated by ethnic Slavs. In the current study two patients with a PALB2 mutation were diagnosed with bilateral breast cancer and they were diagnosed with the third primary cancer. One of these had esophageal cancer and the other had bladder cancer. In previous studies, three male breast cancer patients with a PALB2 mutation had a second primary cancer (thyroid cancer, melanoma, prostate) [16,21]. Male breast cancer patients who carry a mutation in PALB2 gene may be candidates for screening for second primary malignancies.
We have previously reported relatively poor survival for women with breast cancer and a PALB2 mutation [30]. Female breast cancer patients who carried one of the two Polish PALB2 founder mutations had a 10-year survival of 48%, compared to 75% for patients with breast cancer without a PALB2 mutation (HR = 2.27, 95% CI 1.64-3.15; p < 0.0001). Studies from Finland and China also reported poor survival of PALB2-associated breast cancer patients [57,58]. In our cohort, 3 of 4 men with breast cancer and a PALB2 mutation died of metastatic cancer. The first patient was diagnosed with bilateral breast cancer at age 52 and died at age 72 of esophageal cancer. The second was diagnosed with unilateral breast cancer at age 42 and died of metastatic breast cancer at age 53. The third patient had bladder cancer and bilateral breast cancer at age 77 and died of breast cancer at age 79. The fourth patient was diagnosed with breast cancer at age of 42 (in 2017) and is currently alive. This data suggests poor prognosis in men with breast cancer and a PALB2 mutation but larger studies are necessary.
We observed a correlation between the three truncating mutation in the CHEK2 gene (c.1100delC, c.444 + 1G > A and del5395(ex10-11del)) and male breast cancer in Poland (OR = 2.93: p = 0.02). The 1100delC mutation in CHEK2 has been evaluated in several other studies [59][60][61][62][63] and four of these showed an increased risk of male breast cancer. In Finland, Hallamies at al. reported an odds ratio of 4.5 for men with the 1100delC allele (p = 0.02) [22]. A study in Netherlands confirmed the increased risk (OR: 4.1, p = 0.005) [20]. In the largest study of 715 male breast cancers the odds ratio associated with the 1100delC mutation in the CHEK2 gene was 3.8 (p = 0.002) [21]. The frequency of the c.1100delC allele in CHEK2 gene varies widely in different populations; the frequency is high in Finland (1.1-1.4%) and the Netherlands (1.3-1.6%) but is lower in Poland (0.2%). As a consequence the contribution of this allele to the burden of male breast cancer varies widely from country to country [20,22,56].
We found that the frequency of the 1100delC founder mutation in the CHEK2 gene to be higher in male breast cancer cases (1.8%) than in females with breast cancer (0.6%) [29]. A similar result was obtained by Hallamies at al. in Finland. The observed frequency of this mutation was higher in male breast cancer patients (5.9%) then among female patients (2.0%) [22,64].
We did not find a significant association between the c.470C > T mutation in the CHEK2 gene and the risk of male breast cancer (p = 0.84). However, multiple primary cancers were diagnosed in three of nine carriers of this mutation (33.3%)(rectal cancer, myeloid leukemia and skin squamous cell carcinoma) In a previous study we reported a correlation between the c.470C > T mutation in the CHEK2 gene and multi-organ cancer susceptibility (breast, colon, kidney, prostate and thyroid cancer) with odds ratios ranging between 1.5 and 2.0 [65]. PALB2 and CHEK2 are clear breast cancer susceptibility genes. PALB2 encodes a BRCA2-binding protein that acts as a linker between BRCA1 and BRCA2 to form a BRCA1-associated genome surveillance complex. The complex is essential for the homologous DNA break repair [66]. Homozygous mutations of PALB2 cause Fanconi anemia, a rare recessive chromosomal breakage syndrome characterized by physical abnormalities, bone marrow failure and a high risk of malignancy. Heterozygous carriers of PALB2 mutations are at increased risk of breast and pancreatic cancers [67,68].
CHEK2 is involved in the p53 pathway of DNA damage responses. CHEK2 interacts with many different proteins. Upon ionizing radiation-induced DNA damage, CHEK2 is activated by ataxia telangiectasia mutated (ATM) and is in turn capable of phosphorylating several substrates including Cdc25A, Cdc25C, p53, and BRCA1, leading to cell cycle arrest, apoptosis and DNA repair [69].
There are several limitations to this study. Patients were selected from a registry of breast cancer cases held at the Hereditary Cancer Center in Szczecin which could have over-represented patients with a positive history of breast and ovarian cancer. The molecular analysis was based on recurrent mutations which may underestimate the total mutation frequency.

Conclusions
Our study shows that mutations in the CHEK2 and PALB2 genes are important risk factors for male breast cancer in Poland. We recommend that a simple test for Polish founder mutations in BRCA1, BRCA2, PALB2 and CHEK2 should be offered to all male breast cancer patients in Poland.

Acknowledgments
The authors wish to thank Kluska P, Wiśniowski R, Siolek M, Putresza E who helped in this study.

Availability of data and materials
All data generated or analysed during this study are included in this published article [and its supplementary information files].

Declarations
Ethics approval and consent to participate All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki Declaration and its later amendments. The study was approved by the Ethics Committee of the Pomeranian Medical University in Szczecin (IRB-BN-001/174/05). All individual participants included in the study provided written informed consent. A confidential ID number was assigned for further identification to each participant and to the corresponding data. Both hard and soft copy of the data kept in a safe place.

Consent for publication
Not applicable.

Competing interests
The authors declare that they have no competing interests.