G C A T

T A C G
G C A T
genes
Article
Multi-Gene Mutation Profiling by Targeted Next-Generation
Sequencing in Premenopausal Breast Cancer
Eleni Zografos 1 , Angeliki Andrikopoulou 1 , Alkistis Maria Papatheodoridi 1 , Maria Kaparelou 1 ,
Garyfalia Bletsa 2 , Michalis Liontos 1 , Meletios-Athanasios Dimopoulos 1 and Flora Zagouri 1, *

1 Department of Clinical Therapeutics, Alexandra Hospital, School of Medicine, National and Kapodistrian
University of Athens, 80 Vasilissis Sofias Avenue, 11528 Athens, Greece; [email protected] (E.Z.);
[email protected] (A.A.); [email protected] (A.M.P.); [email protected] (M.K.);
[email protected] (M.L.); [email protected] (M.-A.D.)
2 Hellenic Anticancer Institute, 10680 Athens, Greece; [email protected]
* Correspondence: [email protected]

Abstract: Breast cancer has distinct etiology, prognoses, and clinical outcomes at premenopausal
ages. Determination of the frequency of germline and somatic mutations will refine our under-
standing of the genetic contribution to premenopausal breast cancer susceptibility. We applied a
comprehensive next generation sequencing-based approach to analyze blood and/or tissue sam-
ples of 54 premenopausal breast cancer patients treated in our clinic. Genetic testing results were
descriptively analyzed in correlation with clinicopathological data. In the present study, 42.5% of
premenopausal breast cancer patients tested carried pathogenic mutations in cancer predisposition
genes (CHEK2, BRCA1, TP53, and MUTYH). Germline variants of unknown/uncertain significance
Citation: Zografos, E.;
(VUSs) in eight different cancer susceptibility genes, namely BRCA1, BRCA2, CHEK2, RAD51C,
Andrikopoulou, A.; Papatheodoridi,
A.M.; Kaparelou, M.; Bletsa, G.;
RAD51D, ATM, BRIP1, and PMS2, were also identified in 14 premenopausal patients (35%). Of
Liontos, M.; Dimopoulos, M.-A.; the breast tumors tested, 61.8% harbored pathogenic somatic variants in tumor suppressor genes
Zagouri, F. Multi-Gene Mutation (TP53, NF1, RB), genes involved in DNA repair (BRCA1, BRCA2, ATM, RAD50), cell proliferation
Profiling by Targeted Next- (PTEN, PIK3C FGFR3, AKT1, ROS1, ERBB2, NOTCH1), and cell adhesion (CTNNB1). This descriptive
Generation Sequencing in study employs the powerful NGS technology to highlight the high frequency of premenopausal cases
Premenopausal Breast Cancer. Genes attributable to genetic predisposition. Mutation identification in a larger cohort may further ensure
2022, 13, 1362. https://doi.org/ that these patients receive tailored treatment according to their menopausal status.
10.3390/genes13081362

Academic Editors: Keywords: premenopausal breast cancer; mutation; multigene panel testing; NGS
Carmen Criscitiello, Nicola Fusco
and Umberto Malapelle

Received: 2 July 2022

1. Introduction
Accepted: 26 July 2022
Published: 29 July 2022
Globally, female breast cancer has become the most commonly diagnosed cancer, with
2,261,419 new diagnoses and 684,996 deaths estimated to have occurred in 2020 [1]. Breast
Publisher’s Note: MDPI stays neutral
malignancy is considered a biologically and clinically heterogeneous entity, with several
with regard to jurisdictional claims in
recognized risk factors, some of which are modifiable, while others non modifiable [2].
published maps and institutional affil-
Among the latter ones, the inextricable link between ageing and breast cancer remains
iations.
undisputed since only about 4–6% of cases are diagnosed in women aged younger than 40
years [3,4]. Unfortunately, premenopausal women, especially younger ones, face inferior
survival, in part because they present with more aggressive clinicopathologic features
Copyright: © 2022 by the authors.
and distinct age–incidence molecular profiles, including greater prevalence of endocrine-
Licensee MDPI, Basel, Switzerland. unresponsive tumors, higher histological grade at diagnosis, nodal involvement, and higher
This article is an open access article rates of Her2/neu overexpression [5–8]. Additionally, early identification of breast cancer
distributed under the terms and is more challenging in premenopausal women due to increased breast density, with tumors
conditions of the Creative Commons often being detected at later stages [9]. Naturally, this age group is of particular interest
Attribution (CC BY) license (https:// from a public and patient health perspective, as they represent a socially active subset of
creativecommons.org/licenses/by/ the general population, and thus research efforts are geared towards ensuring the optimal
4.0/). individualized management for those patients.

Genes 2022, 13, 1362. https://doi.org/10.3390/genes13081362 https://www.mdpi.com/journal/genes

Genes 2022, 13, 1362 2 of 14

Regarding the familial predisposition of the disease, genetic risk factors should be con-
sidered more in premenopausal patients, since a higher incidence of pathogenic germline
mutations has been reported in younger women. In this setting, according to the latest
international guidelines, hereditary cancer risk evaluation and testing is clinically indicated
for all female breast cancer patients diagnosed under the age of 45 [10]. These women are,
in their overwhelming majority, premenopausal, as epidemiological data for the timing of
menopause in developed nations estimate a median age ranging from 48–52 years [11,12].
The hereditary component of premenopausal breast cancer is further illustrated by the
considerably higher BRCA1/2 mutation prevalence in families with a history of breast
cancer and premenopausal female members affected by the disease [13]. Prior to 2020, the
National Comprehensive Cancer Network (NCCN) Guidelines for Breast and Ovarian Can-
cer Genetic/Familial High-Risk Assessment mainly focused on testing criteria for BRCA1/2
and determining appropriate risk management for carriers of pathogenic variants in these
two genes [14]. However, cancer predisposing mutations are not limited to BRCA, with
the frequency of pathogenic germline variants in known risk genes in young women being
as high as 23% [15,16]. This newly acquired evidence that strongly highlights the clinical
validity of protein truncating variants in genes beyond BRCA1/2, led to the expansion
of these guidelines to include testing of other high-risk breast cancer genes, including
CHEK2, CDH1, BRIP1, NF1, PALB2, PTEN, RAD51C, RAD51D, STK11, and TP53 [17,18].
The inclusion of these genes in the genetic susceptibility evaluation is based on the results
of either prospective cohort studies in a population-based setting or of multiple traditional
retrospective case-control studies that have demonstrated increased breast cancer risk and
whose findings are collectively supported by a uniform consensus [10].
Nowadays, clinical testing with panels of multiple genes has become easily accessible,
especially with the widespread use of affordable sequencing. In this context, most young
breast cancer patients are offered the option of multigene panel testing, which does not
only include high-penetrance susceptibility genes but can also encompass genes associ-
ated with lower penetrance. Genetic risk evaluation can have a substantial impact on
ensuring the individualized management of this cohort since it can help distinguish DNA
damage response deficient subgroups of patients who may benefit from platinum-based
chemotherapy [19] and poly ADP-ribose polymerase [PARP] inhibition [20]. Additionally,
germline genetic testing offers the potential for establishing targeted follow-up protocols
that will ensure early detection of subsequent tumors that are more frequent in high-risk
gene carriers [21]. Early genetic diagnosis offers a significant benefit not only for the af-
fected patient, but also for at-risk family members to enable genetically targeted disease
prevention (chemoprevention and/or prophylactic surgeries) [22]. It further allows carriers
of pathogenic variants to make decisions regarding family planning, which is an aspect of
great importance for the psychosocial well-being of this age group [23].
Despite the paramount implication of genetic testing and the knowledge of inherited
cancer susceptibility in breast cancer patients with younger age of onset, the genetic
background of the disease in premenopausal women remains an understudied field. In the
present descriptive study, we applied multiple-gene panel testing to cancer susceptibility
genes using next generation sequencing (NGS)-based technology aiming to identify the
proportion of premenopausal breast cancer cases attributable to genetic predisposition
and to assess the prevalence of germline (i.e., inherited) and somatic mutations in women
treated in our clinic prior to their menopause.

2. Materials and Methods

2.1. Patients
In this retrospective cohort study, incident cases of 54 premenopausal breast cancer
patients that underwent Next Generation Sequencing (NGS) in blood samples and/or
paraffin blocks were collected. All women attended the Oncology Department of the
National and Kapodistrian University of Athens, at the “Alexandra” Hospital in Athens,
Greece between January 2019 and December 2021. The sample pool derived from the
Genes 2022, 13, 1362 3 of 14

hospital’s patient database, from which consecutive cases satisfying the abovementioned
inclusion criteria were selected. The study was performed in accordance with the Helsinki
Declaration and was approved by the Institutional Review Board (IRB) of the participating
hospital. Participation was voluntary and, once informed consent was granted by each of
the eligible patients, medical files of the participants were reviewed; researchers collected
demographic and clinical data, including age at diagnosis, family cancer history, genetic
testing results, and histopathologic evaluation (tumor stage, size, grade, lymph node status,
hormone receptor, ki67 expression, and HER2 status). The recommended definition of ER
and PR positivity on the AJCC Staging Manual (8th edition) is 1% or more of cells that
score positive by immunostaining [24]. Finally, a blood sample was collected from each
premenopausal breast cancer patient, along with archival formalin fixed paraffin embedded
(FFPE) breast tissue deriving from either mastectomy or breast conserving surgery prior to
adjuvant treatment, whenever FFPE samples were available.

2.2. DNA Extraction and Targeted Next Generation Sequencing

Concerning germline targeted sequencing, genomic deoxyribonucleic acid (DNA) was
isolated from whole blood samples that were collected in EDTA-containing tubes. Follow-
ing centrifugation for 10 min at 3000 rpm at room temperature, each plasma sample was
collected, given a unique identifier, and stored at −80 ◦ C until use. Isolation of plasma DNA
was performed using the QIAsymphony DSP DNA Mini Kit (Qiagen, Germantown, MD,
USA) and indexed genomic libraries were prepared to target the sequence of 94 cancer pre-
disposing genes using the Trusight™ Comprehensive Hereditary Cancer Panel—Nextera™
DNA Flex Pre-enrichment Library Prep (Illumina, San Diego, CA, USA). Libraries were
qualitatively and quantitatively evaluated using a Fragment Analyzer (Advanced Analyti-
cal Technologies, Heidelberg, Germany) and sequenced on a MiSeq, NextSeq 500, or Hiseq
2500 sequencing platform (Illumina Inc, San Diego, CA, USA), where coding regions of
the tested genes had a minimum base and amplicon coverage of 30× for the 96.2% of the
targeted regions.
For the somatic analysis research, 35 paraffin-embedded breast tissues were cut at
slices of 10 µm thickness. Tumor DNA was isolated from tissue samples using the QIAamp
DNA FFPE Tissue (Qiagen, Germantown, MD, USA) or the Ion Ampliseq Custom Next
Generation Sequencing (NGS) DNA panel (Thermo Fisher Scientific, Waltham, MA, USA),
following the manufacturer’s protocol. Tissues then underwent DNA extraction via the
QIAamp DNA FFPE Tissue, and libraries were constructed employing the AmpliSeq for
Illumina Comprehensive Panel v3, that offers coverage of key cancer-associated genes.
The NGS study was performed using an Illumina platform (MiSeq, NextSeq500 or No-
vaSeq) with a median amplicon coverage of 300× for the 93.4% of the targeted regions. In
cases where DNA extraction was executed via the Ion Ampliseq kit, targeted NGS was
performed using the Ion GeneStudio S5 Prime semiconductor-based system (Thermo Fisher
Scientific, Waltham, MA, USA) with a median amplicon cover of 2000×. Subsequently,
the Ion Reporter™ Software (Thermo Fisher Scientific, Waltham, MA, USA) was used for
streamlined data analysis and variant calling against the human reference genome GRCh38.
Additionally, data were curated manually using the genomic analysis software tool On-
comine Reporter (Thermo Fisher Scientific, Waltham, MA, USA) and the available catalogs
of genomic variation observed in the germline as well as those that appear in tumors
as somatic mutations (dbSNP, COSMIC, ClinVar, gnomAD etc.). All sequenced variants
were filtered and evaluated based on the recommendations published by the American
College of Medical Genetics and Genomics (ACMG) [25] and the NCCN guidelines [26].
More specifically, identified alterations were characterized as pathogenic when classified as
disease causing or as variants of unknown significance (VUS) when evidence regarding
their pathogenicity was either conflicting or limited, according to the criteria recommended
to describe variants identified in Mendelian disorders by ACMG [25].
Genes 2022, 13, 1362 4 of 14

2.3. Statistical Analysis

All analyses were descriptive, according to the methodological features of an obser-
vational study with a non-interventional design. Categorical variables were displayed as
frequency tables (N, %). The statistical analysis was carried out using the SPSS software.

3. Results
3.1. Characteristics of the Study Cohort
Histopathological features of the 54 premenopausal breast tumors are presented
in Table 1. The median age of the patients was 40 years (SD = 6.7). The predominant
histological type among patients enrolled in our study was invasive ductal carcinoma (IDC)
(96.3%). The majority of tumors was of high grade (68.5%), and pathological measurements
regarding tumor size revealed that 48.2% of them were above 2 cm (T2, T3, T4). Concerning
receptor status, 77.8% of premenopausal breast cancer cases were ER positive, 74.1% PR
positive, and 72.2% of them HER2 negative. Of note, 13.0% of the participants’ tumors
were classified as triple receptor-negative breast cancer (TNBC), with the Luminal B, HER2-
negative subtype being the prevalent one in our Greek cohort (35.2%). Axillary node
infiltration was identified in 61.1% of cases. Additionally, 38.9% of our premenopausal
patients reported a positive family history of malignancy, defined as ≥ 1 first- or second-
degree relatives having a recorded cancer diagnosis.

3.2. Germline and Somatic Mutations

Fifty-four premenopausal patients with breast carcinoma that underwent genetic
testing via NGS panels for either germline mutations (40), somatic mutations (34), or both
(20), were included in this study. For those with a positive mutation result from genetic
testing, Table 2 shows the identified germline mutations in cancer predisposition genes and
the exact location of each mutation in the affected gene, along with the somatic mutational
pattern of the premenopausal patients whose tumor samples we were able to obtain.
The detection rate of germline mutations among the cases tested was 42.5% (17/40).
Regarding germline pathogenic variants, CHEK2 was mutated in three out of seventeen
premenopausal women, followed by BRCA1 (2 of 17), TP53 (1 of 17), and MUTYH (1
of 17). Interestingly, none of our subjects were identified to have the same pathogenic
germline mutation. However, several of our premenopausal breast cancer patients carried
germline variants of unknown/uncertain significance (VUSs) in eight different breast
cancer susceptibility genes, namely BRCA1, BRCA2, CHEK2, RAD51C, RAD51D, ATM,
BRIP1, and PMS2. Additionally, five out of the twenty patients tested for both intrinsic and
acquired genomic alterations were carriers of concurrent germline and somatic variants.
The complete list of somatic variants identified in our study cohort, both pathogenic
and of unknown significance, is presented in Table 2. Pathogenic somatic mutations
were identified via NGS in 21 out of the 34 premenopausal breast tumors tested (61.8%).
Notably, TP53 (10 of 21; 47.6%) and PIK3CA (8 of 21; 38.1%) were the genes that most
frequently harbored pathogenic somatic mutations in premenopausal cases, followed by
PTEN (4 of 21), BRCA2 (1 of 21), AKT1 (1 of 21), ATM (1 of 21), and ERBB2 (1 of 21).
Collectively, all germline and somatic mutations of premenopausal women with breast
cancer as determined by NGS in relation to each patient’s distinct clinical characteristics
and histological staging is presented in greater detail in Supplementary Table S1.
Genes 2022, 13, 1362 5 of 14

Table 1. Histopathological Status of Premenopausal Breast Cancer Tumors.

Parameters Numbers (%)

20–29 3 (5.6%)
30–39 20 (37.0%)
Age range
40–49 27 (50.0%)
50–52 4 (7.4%)
IDC 52 (96.3%)
ILC 2 (3.7%)
Histological type
Other (medullary, inflammatory, metaplastic) 0 (0%)
Low (grade I) 2 (3.7%)
Intermediate (grade II) 15 (27.8%)
Tumor grade
High (grade III) 37 (68.5%)
IA 13 (24.1%)
IIA 9 (16.7%)
IIB 8 (14.8%)
Tumor stage IIIA 11 (20.4%)
IIIB 1 (1.9%)
IIIC 7 (13%)
IV 5 (9.3%)
T1 (≤2 cm) 21 (38.9%)
T2 (> 2cm but ≤5 cm) 18 (33.3%)
T3 (>5 cm) 7 (13.0%)
Tumor size (T)
T4 3 (1.9%)
Tx 4 (7.4%)
N/A 1 (1.9%)
Negative 12 (22.2%)
ER Positive 42 (77.8%)
Negative 14 (25.9%)
PR Positive 40 (74.1%)
Negative 39 (72.2%)
HER2 Positive 15 (27.8%)
Luminal A 13 (24.1%)
Luminal B (HER2 negative) 19 (35.2%)
Molecular Subtype Luminal B (HER2 positive) 11 (20.4%)
HER2 enriched 4 (7.4%)
Triple negative 7 (13%)
N0 21 (38.9%)
N1 13 (24.1%)
N2 8 (14.8%)
Lymph Nodes
N3 6 (11.1%)
Nx 5 (9.3%)
N/A 1 (1.9%)
Yes 21 (38.9%)
Family History No 33 (61.1%)
Genes 2022, 13, 1362 6 of 14

Table 2. Germline and Somatic mutations identified in premenopausal breast cancer cases.

Gene with Germline Nucleotide Change Codon Change Somatic Mutation

Clinical Significance Somatic Mutation VUS Patient Number
Mutation (cDNA) (Protein) Pathogenic
c. 5266dup p. Gln1756Profs*74 Pathogenic (−) (−) 13
BRCA1 (Chr17) c. 3649T>C p. Ser1217Pro VUS (−) (−) 29
c. 3700_3704del p. Val1234Glnfs*8 Pathogenic N/A N/A 32
c. 352C>T p. Arg118Cys VUS N/A N/A 32
c. 8386C>T p. Pro2796Ser VUS N/A N/A 34
c. 1342C>T: p. Arg448Cys VUS N/A N/A 30
BRCA2 (Chr13)
c. 9613_ BRCA2 c. 9613_
p. Ala3205Leu VUS (−) 31
9614delinsCT 9614delinsCT
BRCA2 c. 9867T>G
c. 9867T>G p. Phe3289Leu VUS TP53 c. 85_86del 33
ROS1 c. 433A>C
c. 1232G>A p. Trp411* Pathogenic N/A N/A 14
c. 470T>C p. Ile157THr Pathogenic N/A N/A 32
c. 470T>C p. Ile157THr Pathogenic (−) (−) 39
CHEK2 (Chr22)
c. 480A>G p. Ile160Met VUS (−) (−) 36
c. 1175C>T p. Ala392Val VUS N/A N/A 38
c. 190G>A p. Glu64Lys VUS (−) FGFR1 c. 2192_*del 37
ROS1 c. 433A>C
c824G>A p. Cys275Tyr Pathogenic TP53 c. 824G>A 8
RET c. 1684A>T
TP53 KMT2C c. 7826G>A
(Chr17) (−) (−) (−) RB1 c. 1988A>G
TP53 c. 824G>A 17
NOTCH1 c. 2453T>C
(−) (−) (−) TP53 c. 424_433del (−) 3
Genes 2022, 13, 1362 7 of 14

Table 2. Cont.

Gene with Germline Nucleotide Change Codon Change Somatic Mutation

Clinical Significance Somatic Mutation VUS Patient Number
Mutation (cDNA) (Protein) Pathogenic
RAD50 c. 1094G>A
TP53 c. 562_
N/A N/A N/A TP53 c. 488A>G 44
564delInsAA
CCND1 c. 724-2A >C
N/A N/A N/A TP53 c. 586C>T (−) 46
TP53 (−) (−) (−) TP53 c. 743G>A KMT2C c. 4873G>A 7
(Chr17)
TP53 c. 743G>A
(−) (−) (−) ROS1 c. 500G>A 27
PIK3CA c. 3140A>G
MET c. 4090C>T
(−) (−) (−) TP53 c. 990del 9
NF1 c. 563C>A
TP53 c. 818G>T
N/A N/A N/A (−) 49
PIK3CA c. 115G>A
RAD51C
c. 80T>C p. Leu27Pro VUS N/A N/A 54
(Chr17)
BRCA2
RAD51D
c. 412A>C: p. Asn138His VUS (−) c. 9613_ 31
(Chr17)
9614delinsCT
BRIP1 (Chr17) c. 2285G>A p. Arg762His VUS N/A N/A 35
MUTYH (Chr1) c. 452A>G p. Tyr151Cys Pathogenic (−) (−) 13
PMS2 (Chr7) c. 1999G>A p. Glu667Lys VUS (−) (−) 53
PTEN c. 481A>G
AKT1 (−) (−) (−) AKT1 c. 49G>A 16
(Chr14) RAD50 c. 443A>G
c. 7475T>G p. Leu2492Arg VUS N/A N/A 28
ATM (Chr11)
(−) (−) (−) ATM c. 494T > G ATM c. 482A>C 15
Genes 2022, 13, 1362 8 of 14

Table 2. Cont.

Gene with Germline Nucleotide Change Codon Change Somatic Mutation

Clinical Significance Somatic Mutation VUS Patient Number
Mutation (cDNA) (Protein) Pathogenic
ROS1 c. 433A>C
ROS1 N/A N/A N/A (−) 48
(Chr6) NOTCH1 c. 7487_7489del
FGFR3 c. 2272G>A
ROS1 c. 1135C>G
(−) (−) (−) PTEN c. 99_100ins 12
BRAF c. 1159G>A
PTEN
(Chr10) TP53 c. 691del
PTEN c. 923delG
N/A N/A N/A PTEN c. 923delA (−) 55
PIK3CA c. 1633G>A
PIK3CA c. 3140A>G BRCA1 c. 3743C>T
(−) (−) (−) 5
PIK3CA c. 1090G>A TP53 c. 622G>A
N/A N/A N/A PIK3CA c. 1624G>A NF1 c. 7301T>C 41
N/A N/A N/A PIK3CA c. 3140A>G RB1 c. 43G>A 43
TP53 c. 815T>C
N/A N/A N/A PIK3CA c. 3140A>T IDH1 c. 949C>T 45
PIK3CA
(Chr3) CTNNB1 c. 1206del
N/A N/A N/A PIK3CA c. 1624G>A RAD50 c. 2604T>G 50
PIK3CA c. 3140A > G RAD50 c. 980G>A
N/A N/A N/A TP53 c.304A>T 51
ERBB2 c. 2264T>C
NF1 c. 2573C>G
PIK3CA c. 1633G > A
N/A N/A N/A PTEN c. 923delG (−) 55
PTEN c. 923delA
Abbreviations: N/A: not available; Chr: Chromosome.
Genes 2022, 13, 1362 9 of 14

4. Discussion
We here retrospectively identified 54 premenopausal breast cancer patients treated in
our clinic, unselected for family history, ethnicity, or subtype. Of these, 40 patients were
tested via comprehensive targeted next generation sequencing and 42.5% (17/40) of them
were identified as carriers of germline mutations in cancer susceptibility genes. Addition-
ally, somatic mutation analysis in 34 premenopausal breast cancer FFPE samples deriving
from our cohort, revealed that 71.4% (25/34) of tested patients were carriers of somatic vari-
ations in tumor suppressor genes (TP53, NF1, RB), genes involved in DNA repair (BRCA1,
BRCA2, ATM, RAD50), in cell proliferation (PTEN, PIK3CA, FGFR3, AKT1, ROS1, ERBB2,
NOTCH1), and in cell adhesion (CTNNB1). To date, little available data on the frequency
and spectra of germline and somatic mutations is specific to premenopausal women with
breast cancer, despite being important for ascertaining the genomic background of this
younger subset of patients.
The testing of germline mutations alongside somatic alterations is rapidly evolving as
an integral part of precision-medicine therapy of patients with cancer. In fact, research hy-
potheses state that a large fraction of cancer predisposition genes, defined as genes in which
germline mutations confer highly or moderately increased risks of cancer, could be onco-
genic when mutated somatically [27]. In our descriptive study, 25% (5/20) patients tested
for both intrinsic and acquired genomic alterations were carriers of concurrent germline
and somatic variants. This observation raises the inevitable question of whether there are
any associations between inherited breast cancer susceptibility loci and somatic mutations
acquired de novo by breast cancer cells, especially in younger premenopausal patients
where the genetic element is more prominent. Recent evidence suggest links between
common germline risk variants and somatic mutations in genes that confer selective breast
tumor growth advantage [28]. In the effort to understand the interaction of germline and
somatic mutations, one could not fail to mention the infamous “two-hit” cancer hypothesis
first proposed by Knudson in 1971 [29]. According to this approach, the first hit could
be an inherited susceptibility variant via the germinal cells or a nonhereditary somatic
mutation in an important cancer predisposition gene; the second hit occurs in somatic cells.
Consequently, it may take fewer stages for individuals with strong genetic predisposition to
develop breast cancer, in comparison to persons with reduced hereditary risk. On the other
hand, in some cases presence of germline mutations may not be related to tumorigenesis
either due to zygosity-dependent phenotype penetrance or loss of the pathogenic germline
allele somatically [30]. In our cohort, only one patient had a coexisting pathogenic germline
mutation and a somatic second hit, interestingly in the same gene (TP53), but on different
loci. The effect of the simultaneous presence of germline and somatic mutations needs to
be further addressed in a larger cohort of patients.
Especially when it comes to hormone sensitive tumors, significant differences in gene
expression and somatic mutation patterns potentially driven by altered hormone levels
have been described between premenopausal and postmenopausal breast cancer, revealing
a menopausal status-dependent role of certain genes [31]. In total, 47.6% of premenopausal
breast cancer samples carried somatic mutations in TP53 and 38.1% in PIK3CA, the two
most-frequently mutated genes according to the literature [32]. This finding is in line with
a targeted deep sequencing analysis on premenopausal breast cancer patients of Latin
American origin, which also pinpointed TP53 as the most frequently mutated gene, fol-
lowed by PIK3CA [33]. The same study reports a clinical association between somatic TP53
mutations and the HER2-enriched molecular subtype, as it has also been shown by others
either in the premenopausal population [34] or even irrespective of menopausal status [35].
This correlation has been attributed to the fact that p53 mutants can potentially induce
HER2 up-regulation and favor the stabilization of the protein [36]. Most of these somatic
mutations are located in residues corresponding to the DNA-binding domain of TP53 and
result in decreased DNA-binding affinity and gene transactivation, thus playing a role in
the early onset and prognosis of breast cancer [37]. Of the ten exonic mutations observed
in this study, nine are in the DNA-binding domain of the TP53 gene, whereas seven out
Genes 2022, 13, 1362 10 of 14

of ten tumors tested positive for HER2 expression, despite only two premenopausal cases
falling under the HER2/neu subtype. Additionally, “acquired” mutations of the oncogene
PIK3CA identified in tumors of our cohort after the implementation of a custom next gener-
ation sequencing DNA panel are commonly found in Luminal/ER-positive tumors, that
comprise the majority of our cases (77.8%). The benefit of alpelisib in PIK3CA-mutated,
hormone-receptor-positive, HER2-negative advanced breast cancer highlights the increas-
ing clinical importance of PIK3CA testing [38]. Although the indication of PI3Kα-selective
inhibitor and degrader is limited to postmenopausal patients, ongoing clinical trials enroll
postmenopausal women that meet the abovementioned inclusion criteria [39]. So far, re-
ports on the somatic genomic landscape of breast cancer state that PIK3CA mutations are
less frequent in young women when compared with older ones [40]. Whether the level of
PI3K pathway activity correlates etiologically with age and/or menopause status bears
further investigation. The remaining cases displayed a variable array of low-frequency
mutations in distinct combinations (NF1, RB, BRCA2, ATM, RAD50, PTEN, FGFR3, AKT1,
ROS1, ERBB2, NOTCH1, CTNNB1), as previously reported [41]. This observation draws
attention to the fact that oncogenic interactions between genes harboring mutations is not
a “straight-line” process but is largely defined by complex arrays of closely intertwined
molecular networks and pathways.
Regarding pathogenic germline variants in premenopausal women, they were all
detected in genes involved in DNA damage repair signaling, with CHEK2 being mutated
in three of seventeen premenopausal patients, followed by BRCA1 (2 of 17), TP53 (1 of 17),
and MUTYH (1 of 17). (3/17), followed by BRCA1 (2/17), TP53 (1/17), and MUTYH (1/17).
In our premenopausal cohort, the frequency of BRCA1 mutations (11.8%) is similar to the
prevalence identified in young breast cancer subsets by other investigators [42]. Interest-
ingly, it has been shown that premenopausal BRCA-mutated patients with breast cancer
are more likely to have more aggressive disease and face recurrences than postmenopausal
carriers, underlying the fact that menopausal status should be taken into consideration as
a potential prognostic factor in BRCA affected patients [43]. Furthermore, the autosomal
inherited TP53 gene found on chromosome 17p13.1 is an established causative factor for
Li-Fraumeni syndrome [44]. Carriers of this mutation have a high lifetime cumulative risk
of developing multiple malignancies and a strong family history of early-onset cancer. This
is consistent with the hereditary profile of our patient that carried the p.C275Y pathogenic
mutation (also known as c. 824G>A), located in coding exon 7 of TP53. Specifically, she
was diagnosed at the very young age of 32 with HER2-enriched breast cancer and had a
first-degree family history, with a mother affected by uterine sarcoma and a father by hepa-
tocellular carcinoma. However, other studies focusing on the premenopausal population
underline that early-onset breast cancer cannot be attributed to TP53 polymorphisms alone,
although specific mutations occur in this young cohort in higher frequency, without, how-
ever, always affecting p53 transactivation function [45]. Concerning CHEK2, we identified
by a targeted gene sequencing panel three premenopausal patients who carried germline
pathogenic mutations that affect the expression of this key cell cycle checkpoint kinase. Pre-
vious studies note an approximately 4% incidence of CHEK2 *1100delC truncation carriers
among premenopausal breast cancer patients [46]. This high-risk allele is associated with
greater than twofold increase in risk and poor prognosis, but it was not detected in our
cohort. This discrepancy may be due to the fact that the CHEK2 c. 1100delC allele is rarely
identified in breast cancer patients of Greek descent [47]. Pathogenic variants in CHEK2
are associated with an increased risk of estrogen receptor-positive breast cancer, although
the risk decreases significantly with age, making these carriers more likely to develop the
disease prior to menopause [48,49]. Notably, the I157T low-risk allele that was identified in
two of our patients is associated with moderate risk and a more favorable prognosis [50].
Lastly, MUTYH mutations that are known to predispose to recessively inherited colorectal
polyposis and cancer have also been associated with breast cancer susceptibility [51,52],
although there are some contradictory findings published [53]. Our results are consistent
with previous studies that reported these mutations in young women diagnosed with
Genes 2022, 13, 1362 11 of 14

invasive breast cancer who were advised to undergo frequent colonoscopy [16]. All in all,
identification of germline mutations and variants in larger patient cohorts is warranted, as
an optimal approach to unravel the underlying heritability of premenopausal breast cancer.
To perform genetic variation screening in premenopausal women with breast cancer
we employed next generation sequencing (NGS) technologies in the form of multigene
panels. There is a current trend in incorporating these technologies in everyday medical care
to guide treatment choices and adopt an integrated counselling approach for each patient.
However, using these technologies in clinical practice on a regular basis presents a number
of issues, one of which is determining how to interpret variants of unknown/uncertain
significance (VUS) [54,55]. These variations in the genetic sequence, including single
nucleotide polymorphisms or amino acid insertions/deletions, cause doubt for clinicians
on how to properly advise patients since the association with cancer risk is unclear. Our
results further illustrate that genetic testing often yields ambiguous results since 35%
(14/40) of our premenopausal patients were carriers of germline VUSs, one in BRCA1, five
in BRCA2, three in CHEK2, two in RAD51, one in ATM, one in BRIP1, and one in PMS2.
These VUSs are alterations that may not even influence the function of the encoded protein;
hence they should not be used to justify a change in clinical management, but should
be managed based on the cancers present in the family [26]. Since the pathogenicity of
germline variants is based on the currently available epidemiologic and functional data, a
clinical takeaway of our germline sequencing findings is the significance of repeat germline
testing as clinical techniques improve and panel sizes grow, especially for individuals at
high germline risk, such as young premenopausal women.
Concerning the limitations of our analysis, one should not fail to mention the small
sample size, which limits the power and generalizability of our work. Another downside
regarding our methodology is the heterogeneity in the data sets in terms of age, molec-
ular subtype, and other clinicopathological characteristics that made direct comparison
somewhat challenging but not unfeasible, due to the exploratory nature of this descrip-
tive study. Further collection of sufficiently large populations of premenopausal patients
for the initial discovery and the subsequent validation is required to fully determine the
prevalence of germline and somatic variants, particularly low frequency ones. Additionally,
larger case–control studies will be needed not only to refine risks of premenopausal breast
cancer associated with these mutations in cancer predisposition genes, but also to allow
researchers to identify well-established mutational signatures characteristic of this special
population. Lastly, since we employed gene panel testing, that allows for screening of
multiple potentially clinically relevant genes, large genomic rearrangements in the familial
breast and ovarian cancer genes were not reported in our work. However, this analysis
contributes to a limited number of studies that provide information on the frequency of
germline and somatic mutations in cancer predisposition genes found in premenopausal
breast cancer, utilizing the powerful NGS technology.
In conclusion, etiology of premenopausal breast cancer was associated with germline
mutations in 42.5% of cases, with 71.4% of tested patients from our cohort carrying somatic
mutations in known cancer predisposition genes. The data presented in our work, and
similar data obtained from expanded germline panel testing in clinical settings, provide
the framework towards enhancing our understanding of premenopausal breast cancer
etiopathogenesis and establishing connections between germline alterations and cancer
risk in specific populations. Given the high incidence of gene mutations, genetic testing
could eventually benefit not only the treatment of premenopausal breast cancer patients,
but also future prevention and control strategies of secondary malignancies in the affected
individual, as well as guidance of at-risk family members.

Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/genes13081362/s1, Table S1: Germline and somatic mutations
of 54 premenopausal women with breast cancer as determined by NGS in relation to each patient’s
distinct clinicopathological characteristics.
Genes 2022, 13, 1362 12 of 14

Author Contributions: E.Z. curated the data and drafted the original manuscript. A.A., A.M.P.,
M.K. and G.B. contributed to the acquisition and analysis of the data, and substantially revised the
manuscript. M.L., F.Z. and M.-A.D. were responsible for the conception and design of the present
work. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: The study was conducted in accordance with the Declaration
of Helsinki and approved by the Institutional Review Board of Alexandra Hospital, Athens, Greece
(Ethics code: 508/14.07.2020).
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.
Conflicts of Interest: The authors declare the following financial interests/personal relationships
which may be considered as potential competing interests: M.L. has received honoraria from Roche,
Astra Zeneca, Astellas, MSD, Janssen, BMS and IPSEN. F.Z. has received honoraria from Astra Zeneca,
Daiichi, Eli-Lilly, Merck, Novartis, Pfizer, and Roche. M.A.D. has received honoraria from Amgen,
Bristol-Myers- Squibb, Celgene, Janssen, and Takeda. The remaining authors declare no conflict
of interest.

References
1. Sung, H.; Ferlay, J.; Siegel, R.L.; Laversanne, M.; Soerjomataram, I.; Jemal, A.; Bray, F. Global Cancer Statistics 2020: GLOBOCAN
Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA. Cancer J. Clin. 2021, 71, 209–249. [CrossRef]
[PubMed]
2. Turkoz, F.P.; Solak, M.; Petekkaya, I.; Keskin, O.; Kertmen, N.; Sarici, F.; Arik, Z.; Babacan, T.; Ozisik, Y.; Altundag, K. Association
between common risk factors and molecular subtypes in breast cancer patients. Breast 2013, 22, 344–350. [CrossRef] [PubMed]
3. Han, W.; Kang, S.Y. Relationship between age at diagnosis and outcome of premenopausal breast cancer: Age less than 35 years is
a reasonable cut-off for defining young age-onset breast cancer. Breast Cancer Res. Treat. 2010, 119, 193–200. [CrossRef] [PubMed]
4. Cancer Research UK Breast Cancer Incidence (Invasive) Statistics by Age. Available online: https://www.cancerresearchuk.
org/health-professional/cancer-statistics/statistics-by-cancer-type/breast-cancer/incidence-invasive#ref-1 (accessed on 10
December 2021).
5. Han, J.G.; Jiang, Y.D.; Zhang, C.H.; Pang, D.; Zhang, M.; Wang, Y.B.; Xue, W.N.; Sun, Q. Clinicopathologic characteristics and
prognosis of young patients with breast cancer. Breast 2011, 20, 370–372. [CrossRef] [PubMed]
6. Anders, C.K.; Hsu, D.S.; Broadwater, G.; Acharya, C.R.; Foekens, J.A.; Zhang, Y.; Wang, Y.; Marcom, P.K.; Marks, J.R.; Febbo, P.G.;
et al. Young age at diagnosis correlates with worse prognosis and defines a subset of breast cancers with shared patterns of gene
expression. J. Clin. Oncol. 2008, 26, 3324–3330. [CrossRef]
7. Fredholm, H.; Eaker, S.; Frisell, J.; Holmberg, L.; Fredriksson, I.; Lindman, H. Breast Cancer in Young Women: Poor Survival
Despite Intensive Treatment. PLoS ONE 2009, 4, e7695. [CrossRef]
8. Gnerlich, J.L.; Deshpande, A.D.; Jeffe, D.B.; Sweet, A.; White, N.; Margenthaler, J.A. Elevated breast cancer mortality in women
younger than age 40 years compared with older women is attributed to poorer survival in early-stage disease. J. Am. Coll. Surg.
2009, 208, 341–347. [CrossRef]
9. Heer, E.; Harper, A.; Escandor, N.; Sung, H.; McCormack, V.; Fidler-Benaoudia, M.M. Global burden and trends in premenopausal
and postmenopausal breast cancer: A population-based study. Lancet Glob. Health 2020, 8, e1027–e1037. [CrossRef]
10. Daly, M.B.; Pal, T.; Berry, M.P.; Buys, S.S.; Dickson, P.; Domchek, S.M.; Elkhanany, A.; Friedman, S.; Goggins, M.; Hutton, M.L.;
et al. Genetic/Familial High-Risk Assessment: Breast, Ovarian, and Pancreatic, Version 2.2021, NCCN Clinical Practice Guidelines
in Oncology. J. Natl. Compr. Cancer Netw. 2021, 19, 77–102. [CrossRef]
11. Davis, S.R.; Lambrinoudaki, I.; Lumsden, M.; Mishra, G.D.; Pal, L.; Rees, M.; Santoro, N.; Simoncini, T. Menopause. Nat. Rev. Dis.
Prim. 2015, 1, 15004. [CrossRef]
12. Schoenaker, D.A.J.M.; Jackson, C.A.; Rowlands, J.V.; Mishra, G.D. Socioeconomic position, lifestyle factors and age at natural
menopause: A systematic review and meta-analyses of studies across six continents. Int. J. Epidemiol. 2014, 43, 1542–1562.
[CrossRef]
13. Kast, K.; Rhiem, K.; Wappenschmidt, B.; Hahnen, E.; Hauke, J.; Bluemcke, B.; Zarghooni, V.; Herold, N.; Ditsch, N.; Kiechle,
M.; et al. Prevalence of BRCA1/2 germline mutations in 21 401 families with breast and ovarian cancer. J. Med. Genet. 2016, 53,
465–471. [CrossRef]
14. Daly, M.B.; Pilarski, R.; Yurgelun, M.B.; Berry, M.P.; Buys, S.S.; Dickson, P.; Domchek, S.M.; Elkhanany, A.; Friedman, S.; Garber,
J.E.; et al. Genetic/familial high-risk assessment: Breast, ovarian, and pancreatic, version 1.2020 featured updates to the NCCN
guidelines. JNCCN J. Natl. Compr. Cancer Netw. 2020, 18, 380–391. [CrossRef]
15. Copson, E.R.; Maishman, T.C.; Tapper, W.J.; Cutress, R.I.; Greville-Heygate, S.; Altman, D.G.; Eccles, B.; Gerty, S.; Durcan, L.T.;
Jones, L.; et al. Germline BRCA mutation and outcome in young-onset breast cancer (POSH): A prospective cohort study. Lancet.
Oncol. 2018, 19, 169. [CrossRef]
Genes 2022, 13, 1362 13 of 14

16. Rummel, S.K.; Lovejoy, L.; Shriver, C.D.; Ellsworth, R.E. Contribution of germline mutations in cancer predisposition genes to
tumor etiology in young women diagnosed with invasive breast cancer. Breast Cancer Res. Treat. 2017, 164, 593–601. [CrossRef]
17. Lee, K.; Seifert, B.A.; Shimelis, H.; Ghosh, R.; Crowley, S.B.; Carter, N.J.; Doonanco, K.; Foreman, A.K.; Ritter, D.I.; Jimenez, S.; et al.
Clinical Validity Assessment of Genes Frequently Tested on Hereditary Breast and Ovarian Cancer Susceptibility Sequencing
Panels. Genet. Med. 2019, 21, 1497. [CrossRef]
18. Dorling, L.; Carvalho, S.; Allen, J.; González-Neira, A.; Luccarini, C.; Wahlström, C.; Pooley, K.A.; Parsons, M.T.; Fortuno, C.;
Wang, Q.; et al. Breast Cancer Risk Genes—Association Analysis in More than 113,000 Women. N. Engl. J. Med. 2021, 384, 428–439.
[CrossRef]
19. Tutt, A.; Tovey, H.; Cheang, M.C.U.; Kernaghan, S.; Kilburn, L.; Gazinska, P.; Owen, J.; Abraham, J.; Barrett, S.; Barrett-Lee, P.;
et al. Carboplatin in BRCA1/2-mutated and triple-negative breast cancer BRCAness subgroups: The TNT Trial. Nat. Med. 2018,
24, 628–637. [CrossRef]
20. Chopra, N.; Tovey, H.; Pearson, A.; Cutts, R.; Toms, C.; Proszek, P.; Hubank, M.; Dowsett, M.; Dodson, A.; Daley, F.; et al.
Homologous recombination DNA repair deficiency and PARP inhibition activity in primary triple negative breast cancer. Nat.
Commun. 2020, 11, 2662. [CrossRef]
21. Harper, D.M.; Nieminen, P.; Donders, G.; Einstein, M.H.; Garcia, F.; Huh, W.K.; Stoler, M.H.; Glavini, K.; Attley, G.; Limacher, J.M.;
et al. The efficacy and safety of Tipapkinogen Sovacivec therapeutic HPV vaccine in cervical intraepithelial neoplasia grades 2
and 3: Randomized controlled phase II trial with 2.5 years of follow-up. Gynecol. Oncol. 2019, 153, 521–529. [CrossRef]
22. Caswell-Jin, J.L.; Zimmer, A.D.; Stedden, W.; Kingham, K.E.; Zhou, A.Y.; Kurian, A.W. Cascade Genetic Testing of Relatives for
Hereditary Cancer Risk: Results of an Online Initiative. JNCI J. Natl. Cancer Inst. 2019, 111, 95. [CrossRef]
23. Dean, M.; Rauscher, E.A. “It was an Emotional Baby”: Previvors’ Family Planning Decision-Making Styles about Hereditary
Breast and Ovarian Cancer Risk. J. Genet. Couns. 2017, 26, 1301–1313. [CrossRef]
24. Zhu, H.; Doğan, B.E. American Joint Committee on Cancer’s Staging System for Breast Cancer, Eighth Edition: Summary for
Clinicians. Eur. J. Breast Health 2021, 17, 234. [CrossRef]
25. Richards, S.; Aziz, N.; Bale, S.; Bick, D.; Das, S.; Gastier-Foster, J.; Grody, W.W.; Hegde, M.; Lyon, E.; Spector, E.; et al. Standards
and guidelines for the interpretation of sequence variants: A joint consensus recommendation of the American College of Medical
Genetics and Genomics and the Association for Molecular Pathology. Genet. Med. 2015, 17, 405–424. [CrossRef]
26. National Comprehensive Cancer Network NCCN Clinical Practice Guidelines in Oncology. Non-Small Cell Lung Cancer. Version
3.2022. Available online: https://www.nccn.org/professionals/physician_gls/pdf/nscl.pdf (accessed on 7 April 2022).
27. Rahman, N. Realizing the promise of cancer predisposition genes. Nature 2014, 505, 302–308. [CrossRef]
28. Zhu, B.; Mukherjee, A.; Machiela, M.J.; Song, L.; Hua, X.; Shi, J.; Garcia-Closas, M.; Chanock, S.J.; Chatterjee, N. An investigation
of the association of genetic susceptibility risk with somatic mutation burden in breast cancer. Br. J. Cancer 2016, 115, 752–760.
[CrossRef] [PubMed]
29. Knudson, A.G., Jr. Mutation and Cancer: Statistical Study of Retinoblastoma. Proc. Natl. Acad. Sci. USA 1971, 68, 820. [CrossRef]
[PubMed]
30. Jonsson, P.; Bandlamudi, C.; Cheng, M.; Srinivasan, P.; Chavan, S.; Friedman, N.; Rosen, E.; Richards, A.; Bouvier, N.; Selcuklu, S.;
et al. Tumour lineage shapes BRCA-mediated phenotypes. Nature 2019, 571, 576–579. [CrossRef] [PubMed]
31. Liao, S.; Hartmaier, R.J.; McGuire, K.P.; Puhalla, S.L.; Luthra, S.; Chandran, U.R.; Ma, T.; Bhargava, R.; Modugno, F.; Davidson,
N.E.; et al. The molecular landscape of premenopausal breast cancer. Breast Cancer Res. 2015, 17, 1–13. [CrossRef]
32. Koboldt, D.C.; Fulton, R.S.; McLellan, M.D.; Schmidt, H.; Kalicki-Veizer, J.; McMichael, J.F.; Fulton, L.L.; Dooling, D.J.; Ding, L.;
Mardis, E.R.; et al. Comprehensive molecular portraits of human breast tumors. Nature 2012, 490, 61. [CrossRef]
33. Olivier, M.; Bouaoun, L.; Villar, S.; Robitaille, A.; Cahais, V.; Heguy, A.; Byrnes, G.; Le Calvez-Kelm, F.; Torres-Mejía, G.;
Alvarado-Cabrero, I.; et al. Molecular features of premenopausal breast cancers in Latin American women: Pilot results from the
PRECAMA study. PLoS ONE 2019, 14, e0210372. [CrossRef]
34. Rath, M.G.; Masciari, S.; Gelman, R.; Miron, A.; Miron, P.; Foley, K.; Richardson, A.L.; Krop, I.E.; Verselis, S.J.; Dillon, D.A.;
et al. Prevalence of germline TP53 mutations in HER2-positive Breast Cancer Patients. Breast Cancer Res. Treat. 2013, 139, 193.
[CrossRef]
35. Darb-Esfahani, S.; Denkert, C.; Stenzinger, A.; Salat, C.; Sinn, B.; Schem, C.; Endris, V.; Klare, P.; Schmitt, W.; Blohmer, J.U.; et al.
Role of TP53 mutations in triple negative and HER2-positive breast cancer treated with neoadjuvant anthracycline/taxane-based
chemotherapy. Oncotarget 2016, 7, 67686. [CrossRef]
36. Román-Rosales, A.A.; García-Villa, E.; Herrera, L.A.; Gariglio, P.; Díaz-Chávez, J. Mutant p53 gain of function induces HER2
over-expression in cancer cells. BMC Cancer 2018, 18, 709. [CrossRef]
37. Damineni, S.; Rao, V.R.; Kumar, S.; Ravuri, R.R.; Kagitha, S.; Dunna, N.R.; Digumarthi, R.; Satti, V. Germline mutations of TP53
gene in breast cancer. Tumor Biol. 2014, 35, 9219–9227. [CrossRef]
38. Suppan, C.; Graf, R.; Jahn, S.; Zhou, Q.; Klocker, E.V.; Bartsch, R.; Terbuch, A.; Kashofer, K.; Regitnig, P.; Lindenmann, J.; et al.
Sensitive and robust liquid biopsy-based detection of PIK3CA mutations in hormone-receptor-positive metastatic breast cancer
patients. Br. J. Cancer 2022, 126, 456–463. [CrossRef]
39. Rugo, H.S.; Lerebours, F.; Ciruelos, E.; Drullinsky, P.; Ruiz-Borrego, M.; Neven, P.; Park, Y.H.; Prat, A.; Bachelot, T.; Juric, D.;
et al. Alpelisib plus fulvestrant in PIK3CA-mutated, hormone receptor-positive advanced breast cancer after a CDK4/6 inhibitor
(BYLieve): One cohort of a phase 2, multicentre, open-label, non-comparative study. Lancet Oncol. 2021, 22, 489–498. [CrossRef]
Genes 2022, 13, 1362 14 of 14

40. Encinas, G.; Maistro, S.; Pasini, F.S.; Katayama, M.L.H.; Brentani, M.M.; De Bock, G.H.; Folgueira, M.A.A.K. Somatic mutations in
breast and serous ovarian cancer young patients: A systematic review and meta-analysis. Rev. Assoc. Med. Bras. 2015, 61, 474–483.
[CrossRef]
41. Agarwal, D.; Nowak, C.; Zhang, N.R.; Pusztai, L.; Hatzis, C. Functional germline variants as potential co-oncogenes. NPJ Breast
Cancer 2017, 3, 46. [CrossRef]
42. Langston, A.A.; Malone, K.E.; Thompson, J.D.; Daling, J.R.; Ostrander, E.A. BRCA1 Mutations in a Population-Based Sample of
Young Women with Breast Cancer. N. Engl. J. Med. 2009, 334, 137–142. [CrossRef]
43. Carlson, K.; Chung, A.; Mirocha, J.; Donovan, C.; Estrada, S.; Siegel, E.; Giuliano, A.; Amersi, F. Menopausal Status and Outcomes
of BRCA Mutation Carriers with Breast Cancer. Am Surg. 2018, 84, 1584–1588. [CrossRef]
44. Correa, H. Li-Fraumeni Syndrome. J. Pediatr. Genet. 2016, 5, 84–88. [CrossRef]
45. Samuel, N.; Id Said, B.; Guha, T.; Novokmet, A.; Li, W.; Silwal-Pandit, L.; Børrsen-Dale, A.L.; Langerød, A.; Hudson, T.J.; Malkin,
D. Assessment of TP53 Polymorphisms and MDM2 SNP309 in Premenopausal Breast Cancer Risk. Hum. Mutat. 2017, 38, 265–268.
[CrossRef]
46. Schmidt, M.K.; Tollenaar, R.A.E.M.; De Kemp, S.R.; Broeks, A.; Cornelisse, C.J.; Smit, V.T.H.B.M.; Peterse, J.L.; Van Leeuwen,
F.E.; Van’t Veer, L.J. Breast cancer survival and tumor characteristics in premenopausal women carrying the CHEK2*1100delC
germline mutation. J. Clin. Oncol. 2007, 25, 64–69. [CrossRef]
47. Apostolou, P.; Fostira, F.; Papamentzelopoulou, M.; Michelli, M.; Panopoulos, C.; Fountzilas, G.; Konstantopoulou, I.; Voutsinas,
G.E.; Yannoukakos, D. CHEK2 c.1100delC allele is rarely identified in Greek breast cancer cases. Cancer Genet. 2015, 208, 129–134.
[CrossRef]
48. Apostolou, P.; Dellatola, V.; Papadimitriou, C.; Kalfakakou, D.; Fountzilas, E.; Faliakou, E.; Fountzilas, G.; Romanidou, O.;
Konstantopoulou, I.; Fostira, F. CHEK2 Pathogenic Variants in Greek Breast Cancer Patients: Evidence for Strong Associations
with Estrogen Receptor Positivity, Overuse of Risk-Reducing Procedures and Population Founder Effects. Cancers 2021, 13, 2106.
[CrossRef]
49. Hu, C.; Hart, S.N.; Gnanaolivu, R.; Huang, H.; Lee, K.Y.; Na, J.; Gao, C.; Lilyquist, J.; Yadav, S.; Boddicker, N.J.; et al. A
Population-Based Study of Genes Previously Implicated in Breast Cancer. N. Engl. J. Med. 2021, 384, 440–451. [CrossRef]
[PubMed]
50. Muranen, T.A.; Greco, D.; Blomqvist, C.; Aittomäki, K.; Khan, S.; Hogervorst, F.; Verhoef, S.; Pharoah, P.D.P.; Dunning, A.M.;
Shah, M.; et al. Genetic modifiers of CHEK2∗1100delC-associated breast cancer risk. Genet. Med. 2017, 19, 599–603. [CrossRef]
[PubMed]
51. Vogt, S.; Jones, N.; Christian, D.; Engel, C.; Nielsen, M.; Kaufmann, A.; Steinke, V.; Vasen, H.F.; Propping, P.; Sampson, J.R.;
et al. Expanded extracolonic tumor spectrum in MUTYH-associated polyposis. Gastroenterology 2009, 137, 1976–1985. [CrossRef]
[PubMed]
52. Wasielewski, M.; Out, A.A.; Vermeulen, J.; Nielsen, M.; Van Den Ouweland, A.; Tops, C.M.J.; Wijnen, J.T.; Vasen, H.F.A.; Weiss,
M.M.; Klijn, J.G.M.; et al. Increased MUTYH mutation frequency among Dutch families with breast cancer and colorectal cancer.
Breast Cancer Res. Treat. 2010, 124, 635–641. [CrossRef] [PubMed]
53. Out, A.A.; Wasielewski, M.; Huijts, P.E.; Van Minderhout, I.J.; Houwing-Duistermaat, J.J.; Tops, C.M.; Nielsen, M.; Seynaeve, C.;
Wijnen, J.T.; Breuning, M.H.; et al. MUTYH gene variants and breast cancer in a Dutch case-control study. Breast Cancer Res. Treat.
2012, 134, 219–227. [CrossRef]
54. Schulz, W.L.; Tormey, C.A.; Torres, R. Computational Approach to Annotating Variants of Unknown Significance in Clinical Next
Generation Sequencing. Lab. Med. 2015, 46, 285–289. [CrossRef]
55. Richter, S.; Haroun, I.; Graham, T.C.; Eisen, A.; Kiss, A.; Warner, E. Variants of unknown significance in BRCA testing: Impact on
risk perception, worry, prevention and counseling. Ann. Oncol. Off. J. Eur. Soc. Med. Oncol. 2013, 24 (Suppl. S8), viii69–viii74.
[CrossRef]