Breast cancer (BC) is the most common cancer and leading cause of cancer-related mortality among women worldwide. In Europe, approximately 500,000 women are diagnosed with BC annually, and in 2018, BC cases were responsible for a third of all cancer related deaths (about 130,000) [1
]. Most women with breast or ovarian cancer (OC) have a sporadic rather than an inherited cancer. However, the majority of hereditary breast and ovarian cancers (HBOC) are due to highly penetrant germline BRCA
variants, which are inherited in an autosomal-dominant fashion: breast cancer susceptibility gene 1 (BRCA1
) or breast cancer susceptibility gene 2 (BRCA2
). In these patients, there are frequently several generations of women affected with BC (often premenopausal) and, in some families, OC as well. The prevalence of BRCA
variants varies based on a number of factors, including type of cancer and age at diagnosis. For individuals whose ethnicity is associated with higher variant frequency, particularly Ashkenazi Jews, any personal or family history of BC is sufficient to warrant consideration of BRCA
testing. Aside from Ashkenazi Jews, founder variants have also been reported worldwide in populations from the Netherlands, Sweden, Hungary, Iceland, Italy, France, South Africa, Pakistan, Asia, and among French Canadians, Hispanics, and African Americans [2
In a recent study, the incidences of BC and OC were reported to be 72% or 44% in BRCA1
carriers and 69% or 17% in BRCA2
carriers, respectively [6
]. Other BRCA
-associated malignancies such as prostate, male breast and pancreatic cancer may also be observed. Less commonly, BC is due to other hereditary syndromes, such as Li-Fraumeni and Cowden, which are associated with variants in the TP53
genes, respectively [8
]. BC is the most prevalent cancer type and the first cause of death among women in Italy [9
]. International guidelines, in cases of known variants in the family, early-onset or triple-negative cancers and multiple relatives with cancer, suggest referral for genetic counseling [10
]. In recent years, poly(ADP-ribose) polymerase (PARP) inhibitors have been developed that target BRCA
pathogenic variants in various cancer types including breast and ovarian cancers [12
]. Thus, the detection of BRCA
variants has a relevant impact both in cancer prevention and in targeted treatment. Typically, variant screening has been performed among affected women, selected on the basis of young age at diagnosis or family cancer history. The aim of this study is to determine the overall germline BRCA
variant frequency and spectrum in healthy Italian individuals at risk or affected by BC or OC by molecular genetic analysis of regions of BRCA1
2. Materials and Methods
2.1. Patients and Samples
Individuals referring to genetic counseling at the Medical Oncology Division of the S. Maria della Misericordia Hospital (Perugia-Italy) in the years 2010–2016 at risk or with a history of BC or OC were included in the study. This cohort of 363 women/men was selected according to the Italian Medical Oncology Association (AIOM) guidelines [13
] based on age at BC/OC onset, number of cancer cases in I- and II degree relatives, and pathological characteristics of BC. Several genetic risk assessment methods are available to estimate the probability of BRCA
variant in individuals in order to select them for molecular diagnosis [14
]. Genetic testing was performed on all individuals >18 years old selected according to the AIOM guidelines and these criteria do not differ from other jurisdictions in Italy.
Knowledge of pathogenetic mutation in the family
Males affected by breast cancer
Women with breast and ovarian cancer
Women affected by breast cancer <36 years old
Women affected by triple negative breast cancer <60 years old
Women with bilateral breast cancer <50 years old
Women with breast cancer <50 years old AND first degree familiarity of:
We chose, however, to utilize BRCA
PRO software that is based on Bayes’ theorem; this requires data on all first, second and third degree relatives of the family proband and incorporates as prior probabilities incidence rates in the US population, allele variant frequencies and penetrances estimated from studies in families with several BC or OC cases [15
]. For unaffected individuals we utilized the Cuzick–Tyrer model that, developed for the International Breast Intervention Study (IBIS-1), incorporates the assessment of additional hereditary factors, body mass index, menopausal status and hormone replacement therapy use [18
]. We considered it suitable for genetic testing of BRCA
variant individuals with an estimated life-time risk of disease ≥10%. The study was conducted in accordance with Good Clinical Practice and the ethical principles of the Declaration of Helsinki and approved by the S. Maria della Misericordia Ethics Committee (CE, protocol 2207/2010). We obtained written informed consent from all participants. Clinical data such as age at diagnosis, hystotype, grading, stage, tumor invasiveness, and receptor status were gathered.
St Gallen guidelines were used to classify BC subtype, based on receptor status [19
]. Data about a second BC and/or OC or other malignancies and the family cancer history in I and II degree relatives were also collected.
2.2. BRCA1/2 Analysis
Ten milliliters of whole blood mixed with EDTA were collected from each patient. Genomic DNA was extracted from blood using the QIAamp DNA mini kit (Qiagen, Hilden, Germany) and quantified using the Qubit dsDNA BR Assay Kit (ThermoFisher Scientific, M0, Italy). All 23 coding exons of BRCA1 (exons 2 to 24) and 26 coding exons of BRCA2 (exons 2 to 27) were amplified in 33 and 46 amplicons, respectively. The primers were designed to cover all coding exons and adjacent 20 base pair introns. The amplified DNA fragments were sequenced using the BigDyeTerminator v.3.1 cycle sequencing kit (Thermo Fisher Scientific) on a 3500 Genetic analyzer (Applied Biosystems, Foster City, CA, USA). Sequencing chromatograms were analyzed for variant detection using Seqscape software v.2.7 (Applied Biosystems, Foster City, CA, USA). In all cases, samples harboring variants were re-amplified and re-sequenced using the same experimental conditions. All sequences were compared with the BRCA1 (NM_007294.3) and BRCA2 (NM_000059.3) reference sequences for variant detection. To identify gross deletions/insertions not detectable by sequencing on the BRCA1/2 genes, we performed the Multiplex Ligation-dependent Probe Amplification (MLPA) using the SALSA P002 BRCA1 and SALSA P045 BRCA2 MLPA probe mix assays (MRC-Holland, Amsterdam, The Netherlands) according to the manufacturer’s instructions. Coffalyser V9.4 software (MRC-Holland, Amsterdam, The Netherlands) was used to analyze MLPA results.
2.3. Variant Classification
According to the IARC recommendations [20
], we classified genetic variants identified into five classes. To annotate BRCA1/2
variants we used: databases such as Breast Cancer Information Core (BIC) [21
Share (formerly Universal Variant Database) [22
], Leiden Open Variation Database (LOVD) [23
], ClinVar-NCBI Database, and American College of Medical Genetics (ACMG) guidelines [24
Variants not found in these databases were classified on the basis of their characteristics.
All variants with conflicting interpretation results by ClinVar-NCBI Database were considered as VUSs. The classification of variants initially considered as VUS was subjected to regular updates, by reviewing the literature and publicly available databases to the best of our knowledge, and modified accordingly. Frameshift and nonsense VUS leading to a premature stop codon were considered likely-pathogenic-class4 and classified in accordance with the ACMG guidelines. All variants were reported according to Human Genome Variation Society nomenclature [25
] according to ENIGMA (Evidence-based Network for the Interpretation of Germline Mutant Alleles) consortium rules for variant classification to obtain the most recent information on variant reclassifications.
2.4. Data Collection and Statistical Analysis
Data were collected using a management system that is integrated with the Umbria Cancer Registry application system [26
Descriptive statistics of patients’ characteristics and sequencing results were presented as median and range for continuous data and as natural frequencies and percentages for categorical data. Pearson Chi-square test or an appropriate Fisher Exact test were used to compare tabular proportions. All data analyses were performed using R software version 3.4.2 (R Foundation for Statistical Computing, Vienna, Austria).
2.5. Immunohistochemistry Analysis of Breast Tumor Samples
Tumor immunohistochemical (IHC) analysis was performed for estrogen receptor (ER) (clone 1D5 diluted 1:15), progesterone receptor (PgR) (clone 1A6 diluted 1:15), and Ki-67 (clone MIB1 diluted 1:15) using the automated platform Bond III (Leica Biosystem, MI, Italy). IHC analysis for evaluation of human epidermal growth factor receptor 2 (HER2) status was performed using the HercepTestTM kit (Dako, Glostrup, Denmark) with an automated system (Autostainer Link 48, Dako) according to the manufacturer’s instructions. HER2 status was defined as negative (HercepTest scores of 0 or 1 +), doubtful (2 + score), and positive (3 ± score). To confirm HER2 status when IHC results were doubtful, we used Fluorescence in-situ hybridization test using a HER2 FISH PharmDxTM kit (Dako Glostrup, Denmark), and gene amplification was recorded when the HER2/centromeric probe for chromosome 17 signal ratio was ≥2.0.
This is a Central Italian study evaluating the prevalence and spectrum of BRCA1/2
variants. We focused our study on variant detection rates and genetic characteristics associated with specific selection criteria for BRCA1/2
testing in high-risk families and patients affected by breast cancer, whereas other authors evaluated clinical implications and strategy of surveillance of women at high risk. Thirteen percent of the individuals evaluated were carriers of a pathogenic variant, according to the range shown in other countries [27
], excluding Ashkenazi Jewish ancestry in which founder variants were prevalent [31
]. The incidence of BRCA1
variants was 7.7% and 6.3%, respectively. According to the literature, we report an incidence of TNBC in BRCA
-carriers (36.4%) about 2-fold higher than that found in sporadic breast cancer. TNBC has been reported to account for 12–24% of all BCs and is associated with an hereditary disease cause [32
]. Approximately 70% of BCs found in BRCA1
variant carriers and up to 23% of BCs in BRCA2
carriers are triple-negative [34
]. Therefore, according to national and international guidelines, women with TNBC diagnosed at an age ≤50–60 years, irrespective of a positive cancer family history, are eligible for germline BRCA
]. As reported in the literature [35
-mutated BC patients showed a significant number of triple-negative cancers (p
< 0.001) and higher Ki-67 expression (p
= 0.008) than in other patients (Table 3
), which represents the higher aggressiveness of the disease. BRCA1
pathogenic/likely pathogenic variants reported in our study were higher than BRCA2
variants (54.9% and 45.0%, respectively). More than 2000 different variants have been identified in BRCA1/2
genes and in some populations, founder variants are the most prevalent ones. For example, up to 2.5% of the general Ashkenazi Jewish population will harbor variants in BRCA1
c.68_69delAG (also known as 185delAG), c.5266dupC (also known as 5382insC) or BRCA2
c.5946delT (also known as 6174delT) [37
We observed 30 distinct pathogenic/likely pathogenic BRCA
variants (14 in BRCA1
and 16 in BRCA2
) and while 23 were observed only once, 5 in BRCA1
and 2 in BRCA2
variants were detected at least two or more times. These seven variants were detected in 23.3% of all the patients with pathogenic BRCA
variant and almost all of them were observed in exon 20 of BRCA1
and exon 11 of BRCA2.
It is important to screen individual populations and ethnic groups to evaluate the true prevalence of BRCA
germline variants [38
], as the frequency and type of BRCA
variants vary significantly depending on ethnicity and race. To our knowledge, our BRCA
study on an Italian population (breast/ovarian cancer patients and healthy population) showed that when several recurrent pathogenic variants are detected, these may be considered as founder variants for this population. If confirmed by further studies, this could have significant implications for preventive population screening and targeted treatments with PARP inhibitors. In our cohort, the BRCA1
c.5266dupC (also known as 5382dupC), considered the founder variant of North-Eastern European origin, was the most frequent, representing 23% of BRCA1
variant carriers, as reported in a previous Italian study [39
In our study, of the 30 pathogenic-likely pathogenic variants observed, 2 (6.6%) are novel and it will be necessary to evaluate their level of penetration in carrier families.
Moreover, different BRCA
variants lead to protein alterations that could have a different impact on the risk of developing tumors in BRCA
variant carriers [40
If a high risk BRCA
variant should be detected, it is important to perform genetic counselling to guide patients and their families regarding risk reduction options and treatment. In our study, we have reported a list of the VUS identified (mostly missense variants) and we note a lack of consensus about their biological/clinical significance among the different databases. Based on the frequency or the co-occurrence of pathogenic variants of these VUS, found in the small number of cases tested in our center, it was not possible to classify these variants. Even though clinician’s decisions cannot be made based on VUS, some of our findings are worthy of attention and deserve further investigation. This is the case, for example, of the young patient (39 years old) with the variant c.4928T > C reported in BRCA2
). Segregation analysis and functional studies should be further performed in this family due to the absence of consensus among databases. Moreover, other breast/ovarian cancer predisposition genes (already present in commercial panels) should also be investigated by next-generation sequencing.
A strength of our study is that it considers not only the affected individuals but also healthy people considered at risk on the basis of the Cuzick–Tyrer program (life-time risk cut off: 10%). Indeed, studies evaluating only patients affected might lead to an overestimate of probability of detecting a variant.
A possible limitation of our study is the selection of individuals for testing. Women should probably not be selected for BRCA testing using only protocols based on risk evaluation tools and strict probability thresholds. Furthermore, there are several different tools to evaluate BRCA risk, and we do not know which is best. Of course, programs with a proactive approach of genetic counseling probably need to enforce rigid selection criteria based on probability threshold in order to contain costs and safeguard their feasibility and ethical sustainability. Besides the variant risk, a woman’s personal motivation and the potential utility of test results for the family should be considered. Another limitation of our study is the absence of segregation analysis within family members that could facilitate follow up of people at high risk of disease and their relatives.
Notwithstanding these limitations, our study provides the identification of patients with heterozygous variants of both BRCA1 and BRCA2, along with individuals carrying one variant and a VUS, underlining the necessity of complete BRCA1/2 testing, which should be offered to all eligible individuals.
The increase of genetic testing leads to the probability of having an non-informative result or VUS. For the management of VUS, it is important to evaluate family history, clinical factors and functional studies on BRCA protein.
Because this information can be confusing and anxiety-provoking to patients, international collaborative efforts are strongly encouraged to ensure that data pertaining to VUS are publicly available.