OF BRCA-ASSOCIATED BREAST CANCER IN THE POPULATION THE RUSSIAN

and it stretches beyond the mutations considered by the "standard" diagnostic panels. Analysis of the entire coding regions of BRCA1 and BRCA2 genes allows increasing efficiency of detection of germline mutations in breast cancer patients at least twofold.

ОРИГИНАЛЬНОЕ ИССЛЕДОВАНИЕ МЕДИЦИНСКАЯ ГЕНЕТИКА ВЕСТНИК РГМУ 1, 2021 VESTNIKRGMU.RU | | cell cycle, regulate transcription and apoptosis, maintain genomic stability [4]. Damage to these genes increases the likelihood of development of cancer, with the majority of such progressions registered in young patients [5,6]. The mutations registered in genes BRCA1 and BRCA2 largely determine the choice of therapy and preventive measures [7]. The current approach to diagnosing hereditary forms of BC adopted in the Russian Federation implies using "standard" diagnostic panels that, relying on PCR, make detection of the BRCA1 and BRCA2 gene mutations most common in our population quick and relatively inexpensive [8]. However, a number of studies points to other clinically significant mutations that cannot be detected with the "standard" panel but increase the risk of cancer development. Therefore, their presence requires a specialized approach in the treatment and prevention of diseases [9].
This research effort aimed to study the features of BRCAassociated breast cancer in the population of the Russian Federation.

METHODS
The study included 4440 patients who underwent examination and treatment at the Russian Scientific Center of Roentgenoradiology from 2010 to 2019. The inclusion criteria were: any age; diagnosed BC. The exclusion criterion was patient's refusal to participate in the study. The age of cancer onset varied from 20 to 90 years ( Table 1). Samples of the tumors of all patients were subjected to histological examination and immunohistochemical analysis (IHC). Compiling the patients' medical histories, we paid special attention to the signals of possible hereditary nature of the disease.
Based on the medical histories and following recommendations of the US National Comprehensive Cancer Network (NCCN) [7], we formed a high-risk group exhibiting clinical signs of hereditary disease (CSHD). The group included 1026 breast cancer patients aged 20-90 years. The patient was added to the high-risk group if she had at least one CSHD: disease manifestation at any age under 50, multiple primary tumors (BC and/or ovarian cancer (OC)), cancer in the family history (BC and/or OC in first-and/or second-degree relatives), triple negative molecular subtype of the tumor.
At the first stage of the study, we employed real-time PCR (RT PCR) in search of the BRCA1 and BRCA2 mutations most common in the Russian Federation: 185delAG, 4153delA, 5382insC, 3819delGTAAA, 3875delGTCT, 300T>G, 2080delA (BRCA1) and 6174delT (BRCA2). All 4440 patients participating in the study were examined. For DNA isolation, we used the M-Sorb kits (Syntol; Russia). OncoGenetics BRCA reagent panel (DNA-Technology; Russia), which includes specific primers for detection of the eight studied mutations, was used to carry out RT PCR.
At the second stage, we examined 290 patients from the high-risk BC development group that had no "standard" mutations detected at the first stage of the study. The entire coding regions of their BRCA1 and BRCA2 genes were analyzed using next generation sequencing (NGS).
Using QIAamp DNA Blood Mini Kit reagents (Qiagen; Germany) and relying on the protocol suggested by the manufacturer, we isolated genomic DNA from peripheral blood. The minimal acceptable DNA concentration was 10 ng/μL. TruSight Cancer panel (Illumina; USA) and TruSight Rapid Capture reagent kit (Illumina; USA) allowed us to prepare the sequencing libraries. We followed manufacturer's instructions and used the selective DNA region capture method.
The prepared libraries were pair-end sequenced (2 × 151 base pairs) on a MiSeq system (Illumina; USA) using MiSeq Reagent Kits v2 (Illumina; USA). The average coverage of the target DNA regions was 100× and over.
The sequencing data were processed with the help of the standard MiSeq Reporter v2.5 (Illumina; USA) software. In some samples, the regions studied presented genetic abnormalities. To increase accuracy, we excluded poor quality sequencing reads from the analysis. Variant Studio 2.2 (Illumina; USA) software was used to annotate and classify the identified sequence variants.
Assessing the clinical significance of the genetic abnormalities identified, we relied on the sequence variant pathogenicity criteria suggested by the American College of Medical Genetics and Genomics (ACMG) [10]  The identified nucleotide sequence changes were verified with the help of the Sanger sequencing method. The analysis was enabled by the ABI PRISM 3100 automated capillary electrophoresis system (Applied Biosystems; USA).

RESULTS
Out of the total sample of RT PCR-diagnosed BC patients (n = 4440), 169 people (3.8%) had BRCA1 and BRCA2 gene mutations detectable with the "standard" diagnostic panels ( Table 2). In the CSHD group, the share of patients with such "standard" gene mutations was 4 times higher: the analysis put it at 15.4%. The most common mutation was 5382insC in the BRCA1 gene. In the overall sample, this variant was detected in 2.9% of patients, while for the high-risk CSHD group this figure was 11.5%, i.e., every 9th patient had the said mutation. Among the identified "standard" mutations, the 5382insC variant was found in 75% of cases. The remaining genetic variants included in the "standard" diagnostic panel were detected at least an order of magnitude less frequently ( Table 2).
Next generation sequencing of the entire coding regions, as well as the BRCA1 and BRCA2 splicing regions, revealed 33 clinically significant variants in 40 out of 290 (13.8%) BC patients from the high-risk group. In 18 cases, the abnormalities were in the BRCA1 gene: nine variants of nonsense mutations, three variants of frameshift deletions, and two abnormalities in splice sites. BRCA2 pathogenic sequence variants were found in 22 patients; there were seven nonsense mutations, eight variants of frameshift deletions and insertions, and two splice site abnormalities (Table 3).
Among the identified genetic disorders, the most common abnormal nucleotide sequence change was the c.3607C>T mutation in BRCA1 (7.5% of cases, three patients). The following pathogenic mutations were detected in approximately 5% of cases: c.4689C>G and c.5224C>T in BRCA1, c.1301_1304delAAAG, c.9089_9090insA and c.3283C>T in BRCA2.
With the exception of mutation 5382insC in BRCA1, the frequency of occurrence of each pathogenic variant detected through NGS is comparable to the frequency of "standard" BULLETIN OF RSMU 1, 2021 VESTNIKRGMU.RU | |  Invasive lobular carcinoma, people (%) 577 (13) Other, people (%) 533 (12) Taking into account the available information on the features of BRCA-associated breast cancer, we analyzed some clinical characteristics of the patients that carried BRCA1 and BRCA2 gene mutations, and studied morphological features of their tumor samples (Table 4). In 94% of patients with BRCA1associated breast cancer and in all patients with BRCA2associated breast cancer, we detected at least one hereditary disease sign (age under 50, cancer in family history, primary multiple tumors, triple negative molecular subtype of the tumor). Six percent of the patients exhibited no clinical signs of a hereditary disease.
Comparison of the groups of patients with identified BRCA1 and BRCA2 gene mutations showed that in the BRCA1associated BC group, the average disease manifestation age was 42 years (20-82 years), and in the BRCA2-associated BC group the onset of the disease was registered at 44, on average (25-79 years old). Moreover, 87% of the BRCA2-associated BC group patients had the cancer diagnosed when they were under 50, and in the BRCA1-associated BC group this figure was 81%.
Over half of gene mutation carriers (63% with abnormal BRCA1 and 74% with mutations in BRCA2) mentioned having blood relatives with BC/OC. In both BRCA1-associated and BRCA2-associated BC groups the frequency of detection of primary multiple malignant neoplasms was rather high (22% and 30% of cases, respectively) ( Table 4).
The examination revealed that the majority of both BRCA1-(91%) and BRCA2-associated tumors (61%) were infiltrating ductal carcinomas (Table 4). However, upon comparison of the groups it was found that the carriers of BRCA1 gene mutations had the said type of cancer in 91% of cases, while those with mutations in BRCA2 -only in 61% (p = 0.0003). For BRCA2associated tumors, on the contrary, there was a predominance of invasive lobular breast cancer (30%) compared with BRCA1associated tumors (5%) (p = 0.0005).
The current classification of molecular subtypes of BC relies on the IHC-enabled detection of expression levels of estrogen (ER), progesterone (PR) and epidermal growth factor (Her2) receptors. These indicators, scored in points, allow classifying the cancer as one of the molecular subtypes, which, in turn, largely determines the disease therapy and prognosis. In the context of this study, we detected triple negative breast cancer ОРИГИНАЛЬНОЕ ИССЛЕДОВАНИЕ МЕДИЦИНСКАЯ ГЕНЕТИКА ВЕСТНИК РГМУ 1, 2021 VESTNIKRGMU.RU | | (TNBC) in 29% of cases (54 patients) in the BRCA1-associated BC group, while for the BRCA2-associated BC group the same figure was only 4% (1 patient). We have also established that almost all BRCA2-associated tumors (96%) were of the luminal subtype and were characterized by the expression of estrogen (ER) and progesterone (PR) receptors (Table 4).

DISCUSSION
The results of this study are consistent with the data of previously published works. We confirmed that in the population of the Russian Federation, BRCA1 and BRCA2 gene mutations are rather frequent, with the most common of them being 5382insC in BRCA1, which is found an order of magnitude more often than other mutations in these genes [2,3,8]. This fact confirms the assumption that this sequence variant is of Slavic origin [2]. Among the rare pathogenic mutations detected with NGS, the most common sequence variant was the c.3607C>T mutation in the BRCA1 gene. This genetic variant was described previously; it is associated with a high risk of development of both BC and OC [11,12].
The analysis of international and Russian publications and databases showed that only a few of the identified rare sequence variants were covered in the Russian studies. The c.3607C>T mutation in the BRCA1 gene was described in a BC patient from St. Petersburg whose family history included cancer patients [13]. The BRCA1 gene mutations c.5224C>T and c.5314C>T were found in the Tatar population in patients with hereditary BC and OC [14]. Mutations c.4689C>G, c.5152+1G>T in BRCA1 and mutations c.6997_6998insT, c.7254_7255delAG and c.658_659delGT in BRCA2 were detected in residents of Siberia and the Far East with hereditary BC and OC [15]. The remaining variants, detected with the help of NGS, were described in foreign publications and databases only.
The results of this study confirm that BC patients with mutations in the BRCA1 and BRCA2 genes often exhibit CSHD, including: disease manifestation at any age under 50, multiple primary tumors (BC and/or OC), cancer in the family history (BC and/or OC in first-and/or second-degree relatives), triple negative molecular subtype of the tumor. The absence of CSHD in 6% of patients with mutations in the BRCA1 may be associated with their unawareness of cancer cases in the family history or development of such mutations de novo.
In conformity with the previously published papers, we found that the average age of cancer onset in the BRCA2associated BC group is 44, which is older than the average onset age registered in the BRCA1-associated BC group (42). According to the results of the combined study that merged the analysis of pathomorphological characteristics of tumors and clinical data of 3797 carriers BRCA1 gene mutations and 2392 patients with abnormal BRCA2 genes, the median age of disease manifestation for the BRCA1-associated BC group was 40, for the BRCA2-associated BC group -43 [16].
The data obtained in this study confirm that the majority of BRCA-associated tumors are infiltrating ductal carcinomas, but among the BRCA2-associated tumors, invasive lobular breast cancer predominates, compared with BRCA1-associated tumors [16].
We have shown that in carriers of the BRCA1 gene mutations (compared to BC patients with this gene undamaged), the tumor is most often characterized by the absence of expression of the estrogen (ER), progesterone (PR) and epidermal growth factor (Her2) receptors, which makes it TNBC [16,17]. Overall, TNBC was diagnosed in 29% of cases in the BRCA1-associated BC group, which is significantly different from the frequency of TNBC recorded in the BRCA2-associated BC group. In context of this study, almost all BRCA2-associated tumors (96%) belonged to the luminal subtype and were characterized by expression of estrogen (ER) and progesterone (PR) receptors, which is also characteristic of sporadic BC [18].

CONCLUSION
Thus, among the residents of the Russian Federation, the range of pathogenic variants of BRCA-associated breast cancer is wide, and it stretches beyond the mutations considered by the "standard" diagnostic panels designed for primary screening. The results of this study highlight the need for analysis of the entire coding regions of BRCA1 and BRCA2 genes, which would allow increasing efficiency of detection of germline mutations in BC patients at least twofold. Due to the certain clinical and morphological features of BRCA-associated breast cancer, such analysis should be prescribed, first of all, for patients in whom the disease manifested at the age under 50 years, whose blood relatives have tumors in their histories (BC and OC), and who have primary multiple malignant neoplasms (BC and BC and/or OC) and triple negative molecular subtype of the tumor.