Screening of BRCA1 and BRCA2 germline mutations in unselected triple‐negative breast cancer patients: A series from north of Morocco

Triple‐negative breast cancer (TNBC) is strongly associated with BRCA1 and BRCA2 germline pathogenic variants. Revised NCCN guidelines recommend that TNBC patients ≤60 years of age should be referred for genetic counseling and consideration of BRCA1/2 testing. The aim of the present study is to characterize germline BRCA1 and BRCA2 variants in a series of TNBC patients from the north of Morocco.

patients with a strong family history of breast and/or ovarian cancer.
Early onset breast cancer and triple-negative breast cancer (TNBC) can also provide an indication for BRCA1/2 testing. TNBC is defined by the lack of expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). It accounted for 12% to 17% of all breast cancer cases, but is over-represented in young women. 1 TNBC is associated with aggressive tumor behavior and a worse prognosis. [1][2][3] It is a highly heterogeneous subgroup, and generally, several genes appear to be involved. The link between TNBC and BRCA1/2 germline pathogenic variants was more widely described in the literature. In this regard, many studies have established the frequency of BRCA1 and/or BRCA2 deleterious variants in TNBC. [4][5][6][7][8][9][10][11][12][13][14][15] This frequency differs according to the populations examined and the selection criteria.
Studies including patients with TNBC selected by age of diagnosis, family history of breast, and/or ovarian cancer or patients referred to genetic counseling have shown a higher rate of germline BRCA1/2 deleterious variants (approximately 50%). 4,[6][7][8] Moreover, a study conducted among an unselected cohort of Ashkenazi women with TNBC have identified BRCA1/2 deleterious variants in around 40% of cases. 9 Although, without any selection criteria, the frequency of BRCA1/2 deleterious variants remains higher among TNBC patients. A study including 1824 TNBC patients unselected for age of diagnosis or family history of breast and/or ovarian cancer showed BRCA1/2 germline pathogenic variants in 11.2% of cases. 5 Several other studies have produced frequencies ranging from 9% to 21%. [10][11][12][13][14][15] The frequency of these pathogenic variants also differs between BRCA1 and BRCA2 genes. In the TNBC group, most of these variants are located in BRCA1. 5,6,[8][9][10]12,13 According to the Consortium of Investigators of Modifiers of BRCA1/2, 68% of tumors arising in BRCA1 carriers were triple negative (TN), compared to only 16% for BRCA2. 16 Currently, several studies recommend to search for BRCA1 and BRCA2 variants in all TNBCs, regardless of age or family history. 5,[10][11][12][13][14][15] Revised National Comprehensive Cancer Network guidelines have considered that TNBC among women ≤60 years old constitutes an additional eligibility criterion for genetic counseling.
In this study, we have screened BRCA1 and BRCA2 genes in a cohort of 32 TNBC patients unselected for family history and age of diagnosis. To the best of our knowledge, this is the first study of its kind in the north of Morocco.

| Pathologic data
ER, PR, and HER2 status were determined using immunohistochemistry (IHC). ER and PR were considered negative when <1% of the tumor cells showed positive nuclear staining. For HER2 staining, we considered as negative IHC scores of 0 and +1, or a score of +2 but HER2 fluorescence in situ hybridization negative.

| Statistical analysis
The comparison between BRCA1/2 deleterious variants carriers and noncarriers was undertaken using the χ 2 test or Fisher's exact test.
A P-value of less than .05 was considered statistically significant.
Statistical analysis was performed using the SPSS 20 software.

| RESULTS
In the current study, 32 female TNBC patients had a BRCA1 and BRCA2 genetic test by next generation sequencing. The age of diagnosis ranged from 28 to 60 years, with an average age of 45 years. All patients had a high mSBR grade (2 or 3). Three patients had a personal and/or family history of breast or ovarian cancer in a first-or second-degree relative.
Sixteen patients had no variant at the coding regions of BRCA1 and BRCA2. Four patients had only benign or likely benign variants.
Four patients had variants of unknown clinical significance (VUS) without other deleterious variants. Finally, pathogenic BRCA1 or BRCA2 variants were found in seven patients with TNBC (21.87%).
All the patients with BRCA1 or BRCA2 pathogenic variants were diagnosed at age ≤60 years. One patient had a medullary carcinoma.
A family history of breast and/or ovarian cancer was not associated with BRCA1/2 deleterious variants in this cohort: only one patient had a family history (a second degree relative). Two patients had a personal history of breast or ovarian cancer (Table 1). No statistically significant differences between BRCA1/2 deleterious variant carriers and noncarriers were observed in clinicopathologic and prognostic characteristics.
Most pathogenic variants were found in BRCA1 (six patients vs one), whereas the VUS were found more in BRCA2, particularly in exon 11 (Figures 1 and 2). In all, six different pathogenic variants Characteristics of TNBC patients with BRCA1/2 pathogenic variants

| Deleterious variants
In

| Variants of unknown clinical significance
In BRCA1, the missense c.

| DISCUSSION
TNBC is strongly associated with BRCA1 and BRCA2 germline deleterious variants. Although, without any selection criteria, the frequency remains higher (9%-21%). 5,[10][11][12][13][14][15] We also found an interesting frequency of deleterious variants of these genes (~22%), particularly in BRCA1 (six vs one). However, the small size of our study reduces the likelihood of finding statistically significant results. This could also explain why there is no significant difference in clinicopathologic and prognostic characteristics between BRCA1/2 pathogenic variant carriers and noncarriers. Despite the small size of our cohort, we believe that this work would help broaden our knowledge of TNBC and BRCA1/2 variants in Morocco.

ACKNOWLEDGMENTS
We would like to thank all the patients who provided samples for the study. We would also like to thank the members of Genetracer Biotech Laboratory, the team of Biomedical Genomics and Oncogenetics Laboratory and the team of Oncology Clinic Al Amal for their help and support. At last, we would like to thank Laraki A. and BaKir M.Y. for their proofreading.

CONFLICT OF INTEREST
The authors declare that they have no competing interests.

AUTHOR CONTRIBUTIONS
All authors contributed to the paper.

ETHICS STATEMENT
Informed consent was obtained from all patients. The Ethics Committee for Biomedical Research in the Faculty of Medicine and Pharmacy of Rabat (CERB) approved this study under number IORG0006594.

CONSENT FOR PUBLICATION
Consent to publish was obtained from all participants.

AVAILABILITY OF DATA AND MATERIAL
The data analyzed during the present study are available from the corresponding author on reasonable request.