Introduction
Approximately 5-10% of all breast cancer (BC) in
women is hereditary BC. Hereditary breast-ovarian
cancer (HBOC) syndrome and Li-Fraumeni syndrome
(LFS) are the most common cause of inherited
BC. HBOC arises from germline mutations in
BRCA1 and BRCA2 genes that are responsible for
approximately half of all cases with hereditary BC.
TP53, PTEN and CDH1 genes are the other highpenetrance
genes that play an important role in the
genetic etiology of BC.[]
TP53 gene is a tumor suppressor gene (also known
as the guardian of the genome). The TP53 protein plays
a major role in response to cell damage. Mutations in
the TP53 gene lead to LFS that has a predisposition to
a wide range of cancers, including BC and childhood
cancers (sarcomas, leukemia, brain tumor, adrenocortical
carcinoma). Women with LSF are at high risk for
developing BC (up to 85% by age 60 years).[] However,
radiotherapy in BC patients with a mutation in
TP53 gene could be a risk factor for developing a second
primary cancer.[]
The most common feature of inherited BC is an
early-onset. Several studies conducted on a mutation
screening in the TP53 gene in patients with BRCA negative
BC, but there are differences in the threshold for
age.[,,] However, patients with LFS have a 25-fold
increased risk of developing cancer under 50 years of
age compared with the general population. For these
reasons, we evaluated 100 patients with BRCA negative
BC under 50 years of age concerning mutation in the
TP53 gene.
In this study, we emphasize the importance of diagnosing
LFS in patients with BRCA negative BC under
50 years of age for patient management, a mutation
screening of asymptomatic kindred and for giving accurate
and reliable genetic counseling.
Methods
Patients
The files of patients with BC who were tested for a mutation
in BRCA genes between 2016 and 2017 years
were reviewed in the Genetic Diagnostic Center. One
hundred patients with BRCA negative BC diagnosed
at age 50 years or younger were selected regardless of
family history. All patients underwent genetic counseling.
It was decided to perform molecular tests in the
TP53 gene.
Ethics committee approval was received for this
study as a retrospective study and informed consent
was obtained from all patients studied.
Isolation of Genomic DNA
Genomic DNA was obtained from all patients by using
the MagPurix Blood DNA Extraction Kit (Zinexts Life
Science Corp., New Taipei City, TAIWAN) according
to the manufacturer"s specifications.
Targeted Next-generation Sequencing (NGS)
NEXTflex® TP53 Amplicon Panel (Bioo Scientific
Corp., Austin, TX, USA) was used for the enrichment
of the coding regions and the intronic regions (up to
the area covered by the kit) of TP53 gene. Targeted NGS
was performed on Illumina MiSeq NGS System (Illumina
Inc., San Diego, CA, USA) using MiSeq Reagent
Kit v2 (500-cycles) (Catalog No: MS-102-2003. Illumina
Inc., San Diego, CA, USA).
NGS Data Analysis
Firstly, "SEQ software" (Genomize, İstanbul, TURKEY)
was used for analyzing the raw data according to the
reference genome of GRCh37. The minimum coverage-
depth was 100X in all target regions. In addition,
Integrative Genomics Viewer (IGV) software was used
for evaluating the reads.[,]
Secondly, variants were detected based on minimum
5X coverage-depth per allele. Then, they were
filtered by the following criteria:
1. Variants that had all submissions as Benign (B)/
Likely Benign (LB) in ClinVar database were excluded,
and
2. Variants that had allele frequency >5% in any population
databases (1000Genomes, ExAC, ESP) were
excluded, and
3. Variants that were in the coding and intronic regions
were included.
Finally, filtered variants were interpreted based on
ACMG Standards and Guidelines recommendations.
[] Ensembl, dbSNP, ClinVar, PubMed, International
Agency for Research on Cancer (IARC) TP53 database,[] LOVD (Leiden Open Variation Database),
HGMD® Professional 2017.3 (Human Gene Mutation
Database) and ExAC, ESP, 1000Genomes population
databases, and in silico prediction tools [-] were
used for interpreting variants.
Confirmation Analysis
The pathogenic variants revealed by the NGS analysis
were confirmed by performing Sanger sequencing on ABI PRISM 3500 DNA analyzer (Applied Biosystems,
Foster City, CA, USA).
Multiplex Ligation-dependent Probe Amplification
Analysis (MLPA)
SALSA® MLPA® P056 TP53 probemix (MRC-Holland,
Amsterdam, the Netherlands) was used for MLPA
analysis in patients who had no pathogenic variants.
The Coffalyser software (MRC-Holland, Amsterdam,
the Netherlands) was used for interpreting the MLPA
data.
Results
Five variants were detected in five of 100 (5%) patients
(Table 1). Four of five variants were assessed as known
as pathogenic/likely pathogenic. These variants were
confirmed by Sanger sequencing. One of five variant
was identified as VUS. All of five variants were found
to be heterozygous. No deletion or duplication was detected
in the TP53 gene in patients who had no pathogenic/
likely pathogenic variant.
Table 1: Classifying the identified variants (n=5)
The mean age at diagnosis for all patients was 39.1
years (range 24-48 years) (Table 2). The mean age of
four patients who had pathogenic/likely pathogenic
variants at diagnosis was 30.2, with a range of 24-38
years.
Table 2: Number of the variants according to age distribution
Discussion
BC is the most common malignancy among women
worldwide. Although most cases of BC are sporadic,
approximately 5-10% of BC cases have a hereditary BC.
The majority of inherited BC in women arises from
mutations in BRCA1 and BRCA2 genes. Other rare
causes of hereditary BC include mutations in the highpenetrance
genes, such as TP53, PTEN, CDH1, STK11,
PALB2, and mismatch repair (MMR) genes.
LFS, also known as SBLA (Sarcoma, Breast,
Leukemia and Adrenal Gland) cancer syndrome, is a
cancer predisposition disease with an autosomal dominant
inheritance. LFS occurs at an early age. LFS arises
from a mutation in the TP53 gene. TP53 gene is activated
when DNA is damaged. Cell cycle progression is
delayed, and DNA is repaired. If TP53 protein is not
activated by mutations, cells with damaged DNA can
survive and proliferate to malignant transformation.
More than 300 germline variants have been reported
in the TP53 gene.[] The majority of variants are missense
variants. Most of them are detected in the DNAbinding
region of the gene (exons 4 to 8).
The clinical spectrum and age in LFS may vary in
populations.[] Patients with TP53 gene mutation
have a high risk of developing a second malignancy.
[] Women with LFS have a high risk of developing
BC with an early age onset. The majority of BC in LFS
occurs between 15 and 44 age of years.[] The lifetime
risk of cancer for women is estimated to be about
90% by 60 years of age.[] Germline molecular testing
(sequencing/MLPA) of TP53 gene is required for
definitive diagnosis of LFS. There are several different
criteria (such as classical LFS, Li-Fraumeni-like (LFL)
syndrome, Chompret criteria) to determine patients
for molecular testing in the TP53 gene. However, not
all patients with TP53 gene mutation meet these clinical
criteria.[]
We identified two pathogenic and two likely pathogenic
variants that were located in the DNA-binding domain
in this study (Fig. 1). All of them had been reported
in IARC TP53 [] and ClinVar databases. One
of the pathogenic variants was found as c.638G>A
(p.Arg213Gln) (Exon6) in patient 2 (Fig. 2). She was
38 years old and diagnosed at 38 years old. Her mother
died at 60 years old because of spinal cord cancer. Her
uncle (maternal) had prostate cancer and died at 65
years old. Her daughter was healthy (18 years old).
The pathogenic variant was also found in her daughter.
The second pathogenic variant was detected in patient
4. It was one of the hotspot mutations as c.817C>T(p.
Arg273Cys) (Exon8) (Fig. 3). She was 42 years old. The
first primary tumor was detected on the right breast at
32 years old, and the second primary tumor was occurred on the left breast at 42 years old. Her father diagnosed
with gastric cancer at 64 years old and died 65
years old. One of the likely pathogenic variants was revealed
in patient 1 as c.469G>T(p.Val157Phe) (Exon5)
(Fig. 4). She was 24 years old and diagnosed at 24 years
old. Her father died because of a brain tumor. She had
three healthy sisters. Her uncle's (paternal) daughter
had a BC. The second likely pathogenic variant was
found in patient 3 as c.332T>C(p.Leu111Pro) (Exon4)
(Fig. 5). She was 37 years old. She diagnosed with BC
at 27 years old. Her father had a brain tumor and died
at 40 years old. Her grandmother (paternal) died at 40
years old because of BC. We also identified a variant
[c.1078G>A(p.Gly360Arg) (Exon10)] as VUS in patient
5, which was reported in ClinVar and IARC databases.
She was 41 years old and diagnosed at 40 years
old. She had not a family history.
Fig 1: Illustration of the TP53 gene showing domains
of the protein together with the location of the
hotspot mutations (shown in red).
*Indicates P/LP variants identified in this study; aa: Amino
acid; P: Pathogenic; LP: Likely pathogenic.
Fig 2: IGV image (a) and electropherogram (b) of patient-2 with a pathogenic variant [TP53:NM_000546:c.638G>A(p.
R213Q)(Exon6) Heterozygous]. The black arrows indicate a variant.
IGV: Integrative genomics viewer.
Fig 3: IGV image (a) and electropherogram (b) of patient-4 with a pathogenic variant [TP53:NM_000546:c.817C>T(p.
Arg273Cys)(Exon8) Heterozygous]. The black arrows indicate a variant.
IGV: Integrative genomics viewer.
Fig 4: IGV image (a) and electropherogram (b) of patient-1 with a likely pathogenic variant [TP53:NM_000546:c.469G>T(p.
Val157Phe)(Exon5) Heterozygous]. The black arrows indicate a variant.
IGV: Integrative genomics viewer.
Fig 5: IGV image (a) and electropherogram (b) of patient-3 with a likely pathogenic variant
[TP53:NM_000546:c.332T>C(p.Leu111Pro) (Exon4) Heterozygous]. The black arrows indicate a variant.
IGV: Integrative genomics viewer.
There are several studies that reported the frequency
of TP53 mutations in patients with BRCA negative
early-onset BC. These studies were conducted in
different ethnic groups with different age thresholds
(Table 3). For example, Lalloo et al.[] carried out a
mutation analysis of the TP53 gene by Sanger sequencing
in 82 English BC patients diagnosed at ?30 age of
years. They identified four variants (4.9%; 4/82). In another
study, Bougeard et al.[] revealed four variants
in 45 French BC patients (0.7%; 4/45) diagnosed at <33
years. The other study was performed by Ginsburg et
al.[] in 95 BC patients diagnosed at <30 age of years
from different ethnicities. They did not find any mutation
in the TP53 gene. Gonzalez et al.[] revealed one
variant in 14 American patients (7%; 1/14) with BC
diagnosed at <30 years. All of the patients had no family
history. In the same study, no mutation was found
in 15 BC patients diagnosed between ages 30 and 49.
However, in our study, we detected two variants in five
BC patients diagnosed at <30 years and two variants in
95 BC patients diagnosed between 30-48 years (Table
2). In another study, Mouchawar et al.[] performed
mutation analyses by Sanger sequencing and by MLPA
method in 41 Australian patients with BC regardless of
family history and diagnosed at <30 years. Two variants
were identified (4.9%; 2/41). One of them was [(p.
Arg175His) (Exon6)] found in a patient who diagnosed
at 24 years old. The other variant was found as a large
deletion encompassing exon 2-4. She was diagnosed
at 26 years old. Lee et al.[] identified five variants in
multi-ethnic 83 Asian patients (6%; 5/83) with BC diagnosed
at ?35 years of age. The median age of onset of
BC was 31 years. In the other study, Rashid et al.[]
detected the frequency of TP53 mutations in 105 Pakistani
patients with BC. Of 67 patients diagnosed at ?30
years of age had no family history. The remaining 38
patients diagnosed at ≤40 years of age had a family history.
Only one variant was revealed in this study (1%;
1/105). It was a frameshift mutation (c.499_500delCA)
in exon 5. It was found in a 28-year-old patient with
no family history. Carraro et al.[] identified only one
variant in 43 Brazilian patients (2.3%; 1/43) with BC
diagnosed ≤35 years of age. It was found as c.427G>A
(p.Val143Met) in a 24 years old patient who had no
family history. Yang et al.[] performed a mutation
analysis of 152 genes associated with hereditary cancers
using NGS in 99 Chinese patients with BC. They
identified three variants in the TP53 gene (3%; 3/99).
Of two variants were found in patients with BC diagnosed
≤30 years of age. They accounted for 10% (2/20)
of all patients with BC diagnosed at ≤30 years in their
study. In another study was performed in the Mexican
population by Gallardo-Alvarado et al.[] They tested
78 Mexican patients with BC diagnosed at <45 years of
age. They determined the frequency of TP53 germline
mutation using NGS as 6.4% (5/78). All five patients
with BC diagnosed before the age of 36 (9.4%; 5/53).
Table 3: Summary of the studies of mutation analysis of the TP53 gene in patients with BRCA negative early-onset BC
BC patients with TP53 mutation and their relatives
who have the same mutation can benefit from surveillance
programs for various cancers to aim at early tumor
detection.[] The surveillance program for BC includes
breast self-examination, clinical examination and imaging.
In general, monthly breast self-examination starting
from the age of 18 years is recommended. Clinical breast examination is recommended every 6-12 months, starting
from the age of 20-25 years. Breast MRI annually is
recommended at 20-75 years. Mammography is controversial
because there are several reports suggesting
that cancers induced by radiation are more common in
patients with LFS.[,] While some of the guidelines
suggest annual breast MRI and mammography, some
guidelines recommend annual breast MRI screening
without mammography.[] Therefore, patients should
be informed about the risks of malignancy associated
with radiotherapy. Risk-reducing mastectomy should be
evaluated in BC patients with TP53 mutation because of
the high contralateral BC risk. Moreover, the option for
risk-reducing bilateral mastectomy should be considered
in women without cancer with TP53 mutation.[]
Recommendations for other cancer risks include comprehensive
physical examinations every 6-12 months,
colonoscopy and upper endoscopy starting at 25 age of
years every 2-5 years,[] dermatological examination especially
for melanoma annually starting at 18 years of
age.[] Whole-body MRI, especially for sarcomas, is
recommended once a year.[,]
Brain MRI is recommended once a year for brain
tumors (can be performed as part of whole-body MRI
or as a separate examination).[]
Conclusion
In conclusion, TP53 gene mutation analyses should
be performed in BRCA-negative BC patients under 50
years of age although differences in the threshold for
age because there is an increased lifetime risk for various
cancers in LFS. TP53 gene mutation analysis can
be performed as a single test or as a part of a multigene
panel using NGS because NGS technology provides simultaneous
analysis of multiple genes in a single test at
a comparable cost to Sanger sequencing.
Peer-review: Externally peer-reviewed.
Conflict of Interest: The authors have no conflicts of interest
to declare.
Ethics Committee Approval: Ethics committee approval
was received for this study as a retrospective study.
Financial Support: The authors declared that this study has
received no financial support.
Authorship contributions: Concept - T.R.Ö., Ö.Ö.K.,
M.E., M.D., G.D.; Design - T.R.Ö., Ö.Ö.K., K.M.E., M.S.G.,
M.E.; Supervision - T.R.Ö., Ö.Ö.K., M.E., M.D., G.D.,
K.M.E., M.S.G., B.Ö., Ö.K., A.K.; Funding - None; Materials
- T.R.Ö., M.E., M.D., G.D.; Data collection and/or processtion Caring
- T.R.Ö., Ö.Ö.K., M.E., M.D., G.D., K.M.E., M.S.G., B.Ö.,
Ö.K., A.K.; Data analysis and/or interpretation - T.R.Ö.,
Ö.Ö.K., K.M.E., M.S.G., B.Ö., Ö.K., A.K.; Literature search -
T.R.Ö., Ö.Ö.K., M.E., M.D., G.D., K.M.E., M.S.G., B.Ö., Ö.K.,
A.K.; Writing - T.R.Ö., Ö.Ö.K., M.E., M.D., G.D., K.M.E.,
M.S.G., B.Ö., Ö.K., A.K.; Critical review - T.R.Ö., Ö.Ö.K.,
M.E., M.D., G.D., K.M.E., M.S.G., B.Ö., Ö.K., A.K.
References
Yang X, Wu J, Lu J, Liu G, Di G, Chen C, et al. Identification
of a comprehensive spectrum of genetic
factors for hereditary breast cancer in a Chinese population
by next-generation sequencing. PLoS One
2015;10(4):e0125571.
Schon K, Tischkowitz M. Clinical implications of
germline mutations in breast cancer: TP53. Breast
Cancer Res Treat 2018;167(2):417-23.
Salmon A, Amikam D, Sodha N, Davidson S, Basel-
Vanagaite L, Eeles RA, et al. Rapid development of
post-radiotherapy sarcoma and breast cancer in a patient
with a novel germline "de-novo" TP53 mutation.
Clin Oncol (R Coll Radiol) 2007;19(7):490-3.
Ginsburg OM, Akbari MR, Aziz Z, Young R, Lynch H,
Ghadirian P, et al. The prevalence of germ-line TP53
mutations in women diagnosed with breast cancer before
age 30. Fam Cancer 2009;8(4):563-7.
Lee DS, Yoon SY, Looi LM, Kang P, Kang IN, Sivanandan
K, et al. Comparable frequency of BRCA1, BRCA2
and TP53 germline mutations in a multi-ethnic Asian
cohort suggests TP53 screening should be offered together
with BRCA1/2 screening to early-onset breast
cancer patients. Breast Cancer Re. 2012;14(2):R66.
Robinson JT, Thorvaldsdóttir H, Winckler W,
Guttman M, Lander ES, Getz G, et al. Integrative genomics
viewer. Nat Biotechnol 2011;29(1):24-6.
Thorvaldsdóttir H, Robinson JT, Mesirov JP. Integrative
Genomics Viewer (IGV): high-performance
genomics data visualization and exploration. Brief
Bioinform 2013;14(2):178-92.
Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster
J, 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(5):405-24.
Bouaoun L, Sonkin D, Ardin M, Hollstein M, Byrnes
G, Zavadil J, et al. TP53 Variations in Human Cancers:
New Lessons from the IARC TP53 Database and Genomics
Data. Hum Mutat 2016;37(9):865-76.
Schwarz JM, Cooper DN, Schuelke M, Seelow D. MutationTaster2:
mutation prediction for the deep-sequencing
age. Nat Methods 2014;11(4):361-2.
Adzhubei IA, Schmidt S, Peshkin L, Ramensky VE,
Gerasimova A, Bork P, et al. A method and server for
predicting damaging missense mutations. Nat Methods
2010;7(4):248-9.
Kumar P, Henikoff S, Ng PC. Predicting the effects of
coding non-synonymous variants on protein function
using the SIFT algorithm. Nat Protoc 2009;4(7):1073-
81.
Desmet FO, Hamroun D, Lalande M, Collod-Béroud
G, Claustres M, Béroud C. Human Splicing Finder: an
online bioinformatics tool to predict splicing signals.
Nucleic Acids Res 2009;37(9):e67.
Lindor NM, McMaster ML, Lindor CJ, Greene MH;
National Cancer Institute, Division of Cancer Prevention,
Community Oncology and Prevention Trials
Research Group. Concise handbook of familial cancer
susceptibility syndromes - second edition. J Natl
Cancer Inst Monogr 2008;(38):1-93.
Rana HQ, Gelman R, LaDuca H, McFarland R, Dalton
E, Thompson J, et al. Differences in TP53 Mutation
Carrier Phenotypes Emerge From Panel-Based
Testing. J Natl Cancer Inst 2018;110(8):863-70.
Bougeard G, Renaux-Petel M, Flaman JM, Charbonnier
C, Fermey P, Belotti M, et al. Revisiting Li-Fraumeni
Syndrome From TP53 Mutation Carriers. J Clin
Oncol 2015;33(21):2345-52.
Mouchawar J, Korch C, Byers T, Pitts TM, Li E,
McCredie MR, et al. Population-based estimate of the
contribution of TP53 mutations to subgroups of earlyonset
breast cancer: Australian Breast Cancer Family
Study. Cancer Res 2010;70(12):4795-800.
Mai PL, Best AF, Peters JA, DeCastro RM, Khincha PP,
Loud JT, et al. Risks of first and subsequent cancers
among TP53 mutation carriers in the National Cancer
Institute Li-Fraumeni syndrome cohort. Cancer
2016;122(23):3673-81.
Lalloo F, Varley J, Moran A, Ellis D, O"dair L, Pharoah
P, et al. BRCA1, BRCA2 and TP53 mutations in very
early-onset breast cancer with associated risks to relatives.
Eur J Cancer 2006;42(8):1143-50.
Bougeard G, Sesboüé R, Baert-Desurmont S, Vasseur
S, Martin C, Tinat J, et al. Molecular basis of the Li-
Fraumeni syndrome: an update from the French LFS
families. J Med Genet 2008;45(8):535-8.
Gonzalez KD, Noltner KA, Buzin CH, Gu D, Wen-
Fong CY, Nguyen VQ, et al. Beyond Li Fraumeni Syndrome:
clinical characteristics of families with p53
germline mutations. J Clin Oncol 2009;27(8):1250?6.
Rashid MU, Gull S, Asghar K, Muhammad N, Amin
A, Hamann U. Prevalence of TP53 germ line mutations
in young Pakistani breast cancer patients. Fam
Cancer 2012;11(2):307-11.
Carraro DM, Koike Folgueira MA, Garcia Lisboa BC,
Ribeiro Olivieri EH, Vitorino Krepischi AC, de Caring valho AF, et al. Comprehensive analysis of BRCA1,
BRCA2 and TP53 germline mutation and tumor characterization:
a portrait of early-onset breast cancer in
Brazil. PLoS One 2013;8(3):e57581.
Gallardo-Alvarado LN, Tusié-Luna MT, Tussié-Luna
MI, Díaz-Chávez J, Segura YX, Bargallo-Rocha E, et al.
Prevalence of germline mutations in the TP53 gene in
patients with early-onset breast cancer in the Mexican
population. BMC Cancer 2019;19(1):118.
Kratz CP, Achatz MI, Brugières L, Frebourg T, Garber
JE, Greer MC, et al. Cancer Screening Recommendations
for Individuals with Li-Fraumeni Syndrome.
Clin Cancer Res 2017;23(11):e38-e45.
Limacher JM, Frebourg T, Natarajan-Ame S, Bergerat
JP. Two metachronous tumors in the radiotherapy
fields of a patient with Li-Fraumeni syndrome. Int J
Cancer 2001;96(4):238-42.
Heymann S, Delaloge S, Rahal A, Caron O, Frebourg
T, Barreau L, et al. Radio-induced malignancies after
breast cancer postoperative radiotherapy in patients
with Li-Fraumeni syndrome. Radiat Oncol 2010;5:104.
Ballinger ML, Best A, Mai PL, Khincha PP, Loud
JT, Peters JA, et al. Baseline Surveillance in Li-Fraumeni
Syndrome Using Whole-Body Magnetic Resonance
Imaging: A Meta-analysis. JAMA Oncol
2017;3(12):1634-9.
