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The Frequency and Management of TP53 Mutation Carriers in Turkish Patients with BRCA-Negative Breast Cancer Under 50 Years of Age

1Health Sciences University İzmir Tepecik Training and Research Hospital, Genetic Diagnostic Center, İzmir-Turkey 2Department of General Surgery, Health Sciences University İzmir Tepecik Training and Research Hospital, İzmir-Turkey 3Department of Medical Oncology, Health Sciences University İzmir Tepecik Training and Research Hospital, İzmir-Turkey 4Department of Radiation Oncology, Health Sciences University İzmir Tepecik Training and Research Hospital, İzmir-Turkey


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. [1] 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). [2] However, radiotherapy in BC patients with a mutation in TP53 gene could be a risk factor for developing a second primary cancer. [3] 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. [2,4,5] 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.

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.

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. [6,7] 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. [8] Ensembl, dbSNP, ClinVar, PubMed, International Agency for Research on Cancer (IARC) TP53 database, [9] LOVD (Leiden Open Variation Database), HGMD® Professional 2017.3 (Human Gene Mutation Database) and ExAC, ESP, 1000Genomes population databases, and in silico prediction tools [10][11][12][13] were used for interpreting variants.

Confirmation Analysis
The pathogenic variants revealed by the NGS analysis were confirmed by performing Sanger sequencing on

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. [14] 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. [15] Patients with TP53 gene mutation have a high risk of developing a second malignancy. [16] 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. [17] The lifetime risk of cancer for women is estimated to be about 90% by 60 years of age. [18] 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. [15] We identified two pathogenic and two likely pathogenic variants that were located in the DNA-binding 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.

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.

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.  [21] 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. [17] 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. [5] 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. [22] 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. [23] 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. [1] 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. [24] 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).
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. [25] 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