The present study retrospectively analyzed a data from a previous study conducted between the years 2007 and 2016 in the MSC Memorial Cancer Centre and Institute of Oncology, Gliwice Branch (COI; Poland), clinicopathological prognostic factors were analyzed in patients with breast cancer with confirmed BRCA1 (n=73), CHEK2 (n=51) and NOD2 (n=31) mutations. The control group was selected from breast cancer patients without mutations (n=392). The patients in all groups (BRCA1, CHEK2 and NOD2 mutation carriers, and the control group) were treated according to the same protocol. All patients provided written informed consent allowing their biological material to be used to clinical research.

All patients were women diagnosed, treated and followed-up at the COI in Gliwice. Patient underwent clinical follow-up examinations every 3 months in the first 2 years, every 6 months thereafter until the 5th year following diagnosis, and every year subsequently. Inclusion criteria were as follows: Breast cancer confirmed by microscopic examination; performance status ZUBROD 0–1; age >18 years; and normal values of renal and liver function, bone marrow. Data for age at onset, menopausal status, surgical procedures, disease stage according to TNM classification (T-the size of the tumor; N-spread of cancer to nearby lymph nodes; and M-metastasis), histology, estrogen and progesterone receptor status, HER2 status and contralateral breast cancer were gathered from hospital records and pathology reports. The analysis of patient medical records was performed according to national legal regulation. The complete characteristics of patients with regard to demographic and clinicopathological features are presented in Tables I and II. Treatment strategies are illustrated in Table III. Preliminary results of this study for BRCA, CHEK2 and NOD2 mutations have been presented in our previous publications (16–18).

The status of CHEK2*1100delC and I157T mutations (GenBank NM_007194.3) was assessed by ASA-PCR and RFLP-PCR techniques, respectively. The present study examined the most common mutations in BRCA1 (c.68_69delAG, c.181T>G, c.4034delA, c.5266dupC and c.3700_3704del5; GenBank NM_007294.3) and BRCA2 (c.5946delT and c.9403delC; GenBank NM_000059.3) present in the Silesian population. The presence of thec.3016_3017insC mutation of NOD2 (GenBank NM_022162.1) was also evaluated in the study group (Table IV). Each patient provided informed consent prior to venous blood collection for a genetic test. Genomic DNA was isolated from peripheral blood leucocytes.

Statistical analysis was performed using STATISTICA 13 software (StatSoft, Inc., Tulsa, OK, USA). The frequency of side effects was counted. The qualitative features are presented as the percentage of their occurrence and were evaluated with Fisher's test and χ² test with the Yates correction. P<0.05 was considered to indicate a statistically significant difference.

BRCA1 mutation carriers were significantly younger in comparison with patients without detected mutations (P=0.0001). The median age of BRCA1 mutation carriers was 43 years (range, 25–74 years) and for the control group it was 53 years (range, 26–78 years). Patients with BRCA-associated breast cancer were also significantly more often in the premenopausal age range compared with the control group (71 vs. 49%; P=0.0005). The median age at menarche was 14 years old, which was similar in the two groups (P=0.559). However, the median number of births was significantly lower in patients with BRCA1 mutations (P=0.0001). The median age of CHEK2 carriers was 50 years (range, 26–71). In the present analysis, CHEK2 carriers were younger, although not significantly, compared with the control group (P=0.081). No significant differences were identified between CHEK2 mutation carriers and the control group according to postmenopausal status (47 vs. 51%; P=0.656). The median age at breast cancer diagnosis for the carriers of the NOD2 mutation was 47 years (range, 27–68). All mutation carriers were younger compared with patients in the control group. The youngest patients were BRCA1 mutation carriers (median age, 43 years), followed by those with the NOD2 mutation (median age, 47 years) and those with the CHEK2 mutation (median age, 50 years) (Table I). There were no differences in age between BRCA1 and NOD2 mutation carriers (P=0.338) or between patients with CHEK2 and NOD2 mutations (P=0.268). BRCA1 mutation carriers were younger in comparison with patients with CHEK2 mutation (P=0.015). BRCA1 mutation carriers were more frequently in the premenopausal period compared with patients with the CHEK2 (P=0.037) or NOD2 mutation (P=0.054).

A history of cancer in the family was reported in 123 (31%) in control group and 80 (52%) patients with mutations (P=0.0001). A family history of cancer was observed more frequently in CHEK2 mutation carriers (61 vs. 31%; P=0.0001) and NOD2 mutation carriers (58 vs. 31%; P=0.005) compared with control group patients. There was a detected tendency towards a family history of cancer in patients with BRCA1 mutations (42 vs. 31%; P=0.065). A family history of breast cancer was reported in patients with mutations in BRCA1 (25 vs. 10%; P=0.0009), CHEK2 (27 vs. 10%; P=0.001) or NOD2 (35 vs. 10%; P=0.0003) in comparison with the control group. Colorectal cancer (14 vs. 5%; P=0.017) and gastric cancer (10 vs. 3%; P=0.044) within the family history were observed more frequently in patients with CHEK2 mutation compared with the control group. Similarly, a significant family history of CNS tumors was identified in patients with CHEK2 mutations (8 vs. 1%; P=0.013) (Table I). There was reported no association between other types of cancer in family history and BRCA1, CHEK2 or NOD2 mutations.

Clinicopathological analysis was conducted. BRCA1-associated cancer had significantly more frequent negative steroid receptor status compared with the control group (62 vs. 31%; P=0.0001). HER2 overexpression was significantly more frequently detected in women without mutations compared with BRCA1 carriers (51 vs. 7%; P=0.0001; Table II, Fig. 1). Histological type G3 was detected more frequently in patients with BRCA1 mutations (55 vs. 31%; P=0.0001; Fig. 2). There were observed differences between two of the analyzed groups (CHEK2 carriers and the control group) with respect to ER-positive status (82 vs. 66%; P=0.001), PR-positive status (78 vs. 59%; P=0.009) and HER2 overexpression (18 vs. 51%; P=0.0001; Table II, Figs. 1, 3 and 4). The histological grade of breast cancer differed between patients with CHEK2 mutations and the control group (12% G3 vs. 31% G3; P=0.003; Table II, Fig. 2). The absence of HER2 overexpression was observed significantly more frequently in NOD2 mutation carriers (90 vs. 49%; P=0.0001) compared with the control group (Table II, Fig. 1). By contrast, there were no differences between NOD2 mutation carriers and the control group with respect to ER (29 vs. 34%; P=0.253) and PR (32 vs. 41%; P=0.447) negative steroid receptor status (Table II, Figs. 3 and 4). A lower histological grade, G1-G2, was observed with similar frequency in NOD2 mutation carriers and the control group (74 vs. 69%; P=0.687; Table II, Fig. 2). G3 tumors were detected in 26% of mutation carriers and 31% of subjects in the control group.

Lymph nodes metastases occurred more frequently in the control group of patients compared with the group with BRCA1 mutations (46 vs. 27%; P=0.004). There was an observed tendency towards the presence of lymph node metastases in patients of the control group compared with CHEK2 mutation carriers (46 vs. 31%; P=0.071). Lymph nodes without metastases (N0) were reported more frequently in patients with NOD2 mutations compared with the control group (74% vs. 54%; P=0.038) (Table II, Fig. 5).

In the present study, patients with BRCA1 mutations had significantly more frequent large tumor sizes (T3-T4) compared with the control group (34 vs. 18%; P=0.002). CHEK2 mutation carriers were slightly more likely to present with locally advanced breast cancer (T3-T4) compared with the control group (25 vs. 18%; P=0.186) and patients with NOD2 mutations exhibited no differences in T grade (Table II, Fig. 6).

The majority of patients in studied groups had the ductal invasive carcinoma subtype: 77% of BRCA1 mutation carriers, 63% of CHEK2 mutation carriers and 87% of patients with NOD2 mutations. The lobular type of breast cancer was detected significantly more frequently in CHEK2 mutation carriers compared with the control group (22 vs. 10%; P=0.033) (Table II).

Compared with the control group, BRCA1 mutation carriers were younger, and more frequently had higher tumor sizes (T3-T4), G3 tumors, negative steroid receptor status (ER-) and tumors without HER2 overexpression. CHEK2 mutation carriers more frequently had tumors without HER2 overexpression, ER-positive receptor status and lower histological grade (G1-G2). Patients with NOD2 mutation were younger and frequently had tumors without HER2 overexpression, when compared with the control group.

The distributions of the molecular types in breast cancer patients with the BRCA1, CHEK2 and NOD2 mutations differed significantly from the distributions of the subtypes in the control group (Table V, Fig. 7). The BRCA1-associated cancers were significantly more often triple negative (TNBC) compared with the tumors in the sporadic cancer cases (62 vs. 14%; P=0.0001). Luminal B subtypes, particularly Luminal B HER2-positive subtypes, were reported more frequently in the control group (56 and 33%, respectively) in comparison with the mutation carriers: BRCA1 (34 and 7%, respectively); CHEK2 (33 and 12%, respectively) and NOD2 (26 and 6%, respectively). Luminal A type breast cancer was diagnosed more frequently in CHEK2 mutation carriers (53%) and NOD2 mutation carriers (45%) compared with the control group (13%) (Table V).