ABSTRACT

Objective:

BRCA1/2 genes play a role in the etiopathogenesis of 10%–30% of triple-negative breast cancer (TNBC). This study aims to investigate the BRCA1/2 genes and the demographic and clinicopathological features in patients with TNBC. The study also examined the impact of cancer history of TNBC individuals’ relatives on the risk of BRCA1/2 mutation carriership rate.

Materials and Methods:

The BRCA1/2 genes of 65 women diagnosed with TNBC between 2011 and 2017 were investigated using next-generation sequencing. We analyzed the correlations of patients’ demographic and clinicopathologic parameters and family history with BRCA1/2 mutation status. We used the χ²-test, t-test, Mann-Whitney U test, and logistic regression statistical methods.

Results:

The BRCA1/2 mutation carrier rate was 16.9%. Patients who had BRCA1/2 mutations were compared with those who did not in terms of demographic and clinicopathological parameters. In the BRCA1/2 mutation carrier group, the Ki-67 index and the number of relatives with cancer were higher than the BRCA1/2 non-carrier group. Logistic regression analysis revealed that when the number of relatives with breast or ovarian cancer was ≥2, the risk of carrying the BRCA1/2 mutation increased by 15-fold. Regardless of the type of cancer (including cancers in other organs besides breast or ovary), the risk of carrying the BRCA1/2 mutation increased 1.3 times with each increase in the number of relatives with cancer for the patient with TNBC.

Conclusion:

In cases with a diagnosis of TNBC, a significant relationship exists between the number of relatives with cancer in the family history and the risk of carrying mutations in the BRCA1/2 genes. This relationship can be confirmed further by large-scale studies with more cases.

Keywords:

BRCA1/2, family history, hereditary cancer, triple-negative breast cancer

Introduction

Breast cancer is the most common type of cancer in females worldwide, and it is the most common cause of cancer-related death in females (1). Triple-negative breast cancer (TNBC) constitutes approximately 12%–24% of breast cancers, highly heterogeneous regarding clinical behavior, morphological features, and genetic background (2). The TNBC subtype is more commonly seen in certain ethnic groups (like African-American, and Hispanic), and BRCA1/2 gene mutation carriers are often diagnosed as intermediate cancer (3). TNBC has a poor prognosis than other breast cancers in terms of relapse rate, frequency of metastasis, and survival parameters. The expression of estrogen receptors (ER) and progesterone receptors (PR) and amplification of HER2 have not been observed in these cancers in analyzes performed using immunohistochemisty. Therefore, endocrine therapies or anti-HER2 therapies cannot be used in these patients (4). Genetic causes play an essential role in the etiopathogenesis of TNBC. The best-known genetic risk factors are BRCA1/2 mutations, which lead to hereditary breast and ovarian cancer syndrome. These genes are involved in controlling the cell cycle, chromatin remodeling, and epigenetic regulation. Loss of function mutations in these tumor suppressor genes, which are important for the continuation of genomic integrity, contribute to initiating carcinogenesis. The prevalence of carrying BRCA1/2 mutations has been reported as 3% in all breast cancer patients and 10%–30% in TNBC patients (5). Approximately 70% of breast cancers are observed in BRCA1 germline mutation carriers, and 16%–23% of breast cancers in BRCA2 germline mutation carriers are the TNBC subtype (6, 7). This single-center cohort study aims to evaluate the relationship between the BRCA1/2 mutation status and demographic characteristics, clinicopathological details, and family cancer histories of 65 patients diagnosed with TNBC.

Materials and Methods

In this study, 65 Turkish female patients with TNBC from the Dr. Abdurrahman Yurtaslan Ankara Oncology Training and Research Hospital, Department of Medical Genetics, were investigated between 2011 and 2017 for their breast cancer’s genetic etiology. BRCA1/2 test standards were applied to the patients in accordance with the National Comprehensive Cancer Network Guidelines (8). They were over 18 years old, and their breast cancers were diagnosed as the primary tumor. The patients’ family history was evaluated by examining at least three generations of pedigree analyses. This study was conducted by considering ethical responsibilities according to the Declaration of Helsinki and approved by the independent Ethics Committee of the Dr. Abdurrahman Yurtaslan Ankara Oncology Training and Research Hospital approved this study (no: 2020-02/536). In this study, all patients were informed about genetic tests and the use of their information, and their consent was obtained.

All patients’ breast cancer was histologically confirmed, and staging was determined based on the sixth edition of the American Joint Committee on Cancer (9). If the percentage of cells stained positive by immunohistochemistry was less than 1%, the ER and PR status was evaluated as negative. For HER2 gene amplification in IHC staining, membranous staining was graded from 0 to +3. Patients with a staining pattern of +2 were evaluated using the fluorescent in situ hybridization method. Those with <2 copies of the HER2 gene were considered negative.

Patients’ genetic analyzes were conducted on the Ion S5™ platform with the next-generation sequencing method and used the Oncomine™ BRCA Research Assay kit. In the analysis, all the coding regions of BRCA1/2 genes and the part containing the 25 base pairs of exon-intron junctions were investigated. The presence of genomic copy number changes in patients was also investigated using a Multiplex ligation-dependent probe amplification method. The Ion Reporter Software Version 5.4 program was used in the bioinformatics analysis of the obtained data. For BRCA1 and BRCA2 genetic analysis, the accession numbers used were NM_007294.3 and NM_000059.3, respectively. Sequence variants were classified using algorithms in the American College of Medical Genetics and Genomics guideline (10).

SPSS (IBM SPSS Statistics 24) program was used in the statistical evaluation of all results. In this study, the c²-test, t-test, Mann-Whitney U test and logistic regression analysis were used, and p<0.05 was considered statistically significant.

Results

The mean age of diagnosis of 65 patients included in this study was 41±10 years. Most patient (73.9%) visits were due to a palpable mass when a cancer diagnosis was made. Of these patients, TNBC was grade 3 in 50%, invasive ductal carcinoma in 80%, and approximately 1-3 cm mass in 72%. The demographic and clinicopathological characteristics of these 65 patients are presented in Table 1. Women who smoked at least ten cigarettes a day for more than ten years were considered positive. In the oral contraceptive usage parameter, patients’ use for five years and longer was positive. Among the relatives of patients in the study group, 20 different types of cancer were detected, including breast, ovarian, endometrium, colon, stomach, liver, biliary tract, lung, laryngeal, bladder, kidney, brain, leukemia, lymphoma, oral, skin, and thyroid cancers. The most common cancers among the relatives were breast, lung, colon, stomach, larynx, prostate, and uterus cancers (Figure 1). In the grouping of relatives diagnosed with cancer, the number of relatives diagnosed with breast and ovarian cancer and the relatives who were diagnosed with any cancer, regardless of the cancer type was determined from pedigree analysis. In our study, the total prevalence of BRCA1/2 mutations evaluated was 16.9% (11/65). Of BRCA1/2 mutations, 63.6% were in BRCA1 (7/11), and 36.4% were in BRCA2 (4/11). The majority of BRCA1 mutations were of the nonsense type, whereas all BRCA2 mutations were of the frameshift type. All mutations were detected in the heterozygous state. Two of these mutations have not been reported before (Table 2). Patients with and without BRCA1/2 pathogenic variants were compared statistically regarding demographic and clinicopathological features (Table 3). In the group with the BRCA1/2 mutation, the Ki-67 index and the number of relatives with cancer were higher than BRCA1/2 mutation-negative group. When other parameters were investigated, no statistically significant difference was found between the groups. The mean Ki-67 index of all patients in the study was 53 (range: 5–100). This Ki-67 index was 78 in the BRCA1/2 mutation carrier group and 40 in BRCA1/2 mutation-negative group. The difference between these two groups was statistically significant (p = 0.022) (Table 4). Logistic regression analysis was performed to investigate the association of BRCA1/2 mutation carrier status and the number of relatives with breast and ovarian cancer. There was a significant relationship between the number of relatives with cancer and BRCA1/2 mutation carrier status (p = 0.006). Those who have two or more relatives with cancer had a 15 times higher risk of carrying a disease-related variant in BRCA1/2 than those without cancer relatives (Table 5). A logistic regression analysis was also performed to investigate the relationship between BRCA1/2 mutation status and all relatives with cancer regardless of the cancer type. In this model, a significant correlation was found (p = 0.047). The risk of BRCA1/2 mutation carrier status means 1.3 times increased risk for cancer among the relatives (Table 6).

Discussion and Conclusion

TNBC is a breast cancer subtype with clinically aggressive behavior and poor prognosis with limited treatment options and poor overall and disease-free survival. This cancer type is extremely heterogeneous regarding clinical, genetic, and histopathologic features. In this cohort study, the mutation status BRCA1/2 and general demographic and clinicopathological features of 65 Turkish women diagnosed with TNBC were investigated to elucidate TNBC’s complex nature. In this study, TNBC’s features were compatible and concordant with the literature regarding parameters such as high Ki-67 index values, diagnosis at an early age and premenopausal period, and mutation status of BRCA1/2 (1, #*#ref2 #*#, 3, 4, 5, 6, 7). In the literature, the BRCA1/2 mutation carrier prevalence of TNBC patients was approximately 10%–30%. In this study, the prevalence of BRCA1/2 mutation carriers was 16.9%, consistent with the literature. The age at diagnosis of the patient group was 40.86±10.28 years; a statistically significant difference was not found between BRCA1/2 mutation non-carriers (40.5) and BRCA1/2 mutation carriers (37). In this study, the number of relatives with breast and ovarian cancer was significantly higher in the BRCA1/2 mutation carrier group than the non-carrier group. Logistic regression analysis showed that TNBC individuals with relatives with two or more breast and ovarian cancer have a 15-fold increase in the BRCA1/2 mutation carrier risk. Studies in the literature have shown that the lifetime risk of breast cancer in females is from 46%–87% in BRCA1 mutation carriers and 38%–84% in BRCA2 mutation carriers. In these studies, it was also observed that the risk of ovarian cancer up to the age of 70 is from 39%–63% in BRCA1 mutation carriers and from 16.5%–27% in BRCA2 mutation carriers (11, 12, 13). In addition to this dramatic increase in risk detected in breast and ovarian cancers, in BRCA1/2 mutation carriers, it has been reported in the literature that there is an increased risk in multiple other cancer types such as pancreas, colon, prostate, buccal cavity, pharynx, kidney, gall bladder, bile duct, cervix, uterine body, bone, stomach, and malignant melanoma. Although BRCA1 and BRCA2 genes both play tumor suppressor roles in the cell, cancers arising from mutations of each of these genes have specific clinicopathological structure. In the literature, these genotype-phenotype correlations were investigated many times. It was determined that observed cancer risks of each of these genes were not the same concerning the age of disease onset, sex, and the primary site of cancer origin (14, 15, 16, 17, 18, 19, 20, 21). The diversity in research findings might be due to many reasons, such as insufficient samples, studies in geographically and ethnically different societies, and diversity in analysis systems.

In this study, relatives had 20 different cancer types based on the pedigree analysis of TNBC patients. Twenty-five of 65 patients had at least one relative with breast cancer. At least one relative of 49 patients had a cancer diagnosis (principally breast, lung, colon, stomach, prostate, other types). Regardless of the type of cancer, a significant relationship was found between the number of relatives diagnosed with cancer and BRCA1/2 mutation carrier status. According to the logistic regression model, it was predicted that the risk of carrying BRCA1/2 mutation would increase 1.3 times with an increase in the number of each relative with cancer for TNBC patients.

This study’s limitations are that the sample group was not large enough, and co-segregation analysis could not be performed for all patients’ relatives. Information about a cancer diagnosis in patients’ relatives was obtained from the pedigree analysis, and pathological reports of most of them could not be reached. Also, environmental factors that may predispose relatives to cancer could not be investigated. In conclusion, TNBC is a heterogeneous cancer type that occurs at an early age, has a poor prognosis, and high histopathologic grade.

In this study, we investigated the relationship between TNBC and BRCA1/2 mutation carrier status of 65 patients. We found a significant relationship between BRCA1/2 mutation carrier status, high Ki-67 index, and the number of relatives with cancer. In the future, further research is needed to determine the importance of these genes in TNBC and help elucidate the complex nature of TNBC.

Key Points

• TNBC type breast cancers are frequently seen in BRCA1/2 genes mutation carriers.

• The Ki67 index of BRCA1/2 gene mutation carriers is high in TNBC.

• In TNBC, the family cancer history is important at the risk of BRCA1/2 carriage.

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