Key Point

• The purpose of this study was to determine the frequency of germline variants in BRCA1, BRCA2, CDH1, PALB2, PTEN and TP53 in a population of Turkish patients admitted to a medical genetics clinic with breast cancer. In addition, the identified variants were assessed in the light of published genetic, surgical and oncological perspectives.

Introduction

Breast cancer is the most prevalent cancer and the leading cause of death among women worldwide, accounting for approximately 12% of all new cancer cases diagnosed in recent years (1). The etiology of breast cancer is multifactorial and complex, and most cases are sporadic, although genetic factors play an important role. The most common cause of hereditary breast cancer is because of inherited germline mutations in the high-penetrant cancer predisposition genes BRCA1 and BRCA2. Beside these genes, advances in DNA sequencing techniques, such as next generation sequencing, have helped to identify additional breast cancer susceptibility genes, including TP53, CDH1, PALB2 and PTEN and various rare gene variants have also been reported to increase the risk of developing breast cancer. The prevalence of BRCA1 and BRCA2 pathogenic variants is estimated to be 1/400 to 1/500 in the general population and is increased in some populations due to the founder effect (2). It is estimated that the risk of developing breast cancer by age of 80 is 72% for BRCA1 mutation carriers and 69% for BRCA2 mutation carriers, respectively. According to the literature, the risk for contralateral breast cancer 20 years after first breast cancer diagnosis is 40% for BRCA1 and 26% for BRCA2 carriers (3).

Tumor protein p53 (TP53) is one of the most mutated genes in cancer, including breast cancer. TP53 is a tumor suppressor gene that encodes tumor protein p53, a transcription factor. Germline TP53 mutations are associated with Li-Fraumeni syndrome, a rare autosomal dominant genetic disorder. In Li-Fraumeni syndrome, in addition to breast cancer, brain tumors, adrenocortical carcinoma, leukemia, and germ cell tumors have been reported. Although germline TP53 mutations are rare and seen in approximately 1% of all breast cancers, the lifetime risk of breast cancer in TP53 mutation carriers is nearly 80–90%, considerably greater than for other genes (4).

The CDH1 gene encodes E-cadherin, a calcium ion-dependent cell-cell adhesion protein, and it is known that germline CDH1 pathogenic variants predispose the individuals to both diffuse gastric cancer and lobular breast cancer (LBC). Studies have shown that estimated cumulative risk of LBC for women is 42% by age 80 years (5, 6). Partner and localizer BRCA2 (PALB2) is one of the important DNA repair genes that co-localizes with BRCA2 in nuclear foci. PALB2 has functions in homologous recombination, recombinational repair and checkpoint mechanisms. Several studies showed the increased risk of breast cancer in PALB2 mutation carriers. In a large cohort, PALB2 mutations were found in approximately 1% of the BRCA1/2 negative patients with breast cancer and in 0.19% of healthy controls (7). Phosphatase and tensin homolog (PTEN) is one of the most common tumor suppressor genes. It is known that germline PTEN mutations predispose to the development of Cowden syndrome, an autosomal dominant inherited cancer syndrome characterized by multiple hamartomas and malignancies, including breast, thyroid, and endometrial cancer. Studies have shown that the prevalence of Cowden syndrome to about 1:200,000 patients and women with Cowden syndrome have a 20% to 50% lifetime risk of developing breast cancer (8).

Patients with suspected hereditary breast cancer should obtain genetic counseling and be informed about the risk factors. The National Comprehensive Cancer Network (NCCN) has published recommendations to guide healthcare providers in identifying individuals with hereditary cancer syndrome (Table 1) (9). In the presence of any of the criteria, there is an indication for genetic testing for hereditary breast cancer as there is high lifetime risk for mutation carriers (Table 2) (10). Detection of germline mutations may lead to the improvement of diagnostics and selection of patients sensitive to targeted therapeutics. Therefore it may help the development of familial screening strategies and may also have important implications for development of specific treatment and prevention protocols in mutation carriers.

Materials and Methods

A total of 195 operated breast cancer patients who attended a Medical Genetics Department between 2019 and 2022 were included. According to the NCCN guideline criteria, in the 195 patients selected for this study, the age and the family history of the patients were the main criteria (Table 1). The study was conducted in accordance with the Declaration of Helsinki and was approved by İzmir Katip Çelebi Non-Interventional Clinical Studies Institutional Review Board (0028/20.01.2022). Medical history was collected from requisition forms and medical records. Written informed consent forms were obtained from all patients. In this study, the exonic regions and exon-intron junctions in BRCA1, BRCA2, CDH1, PALB2, PTEN and TP53 genes were analyzed. The workflow included sample extraction, library preparation, sequencing and bioinformatics steps. In the sample extraction step, DNA from blood samples was extracted using EZ1 DSP DNA Blood Kit (Qiagen, Hilden, Germany). Qubit dsDNA HS, Thermo kit with Qubit 3 Fluorometer were performed to measure and optimize DNA concentration. QIAseq Targeted DNA Human BRCA1 and BRCA2 Plus Panel, Qiagen (333505) were used according to manufacturer instructions for Illumina sequencers. The genomic DNA was fragmented and all fragmented DNA was barcoded with unique molecular indices to track the original DNA molecule; hence, high sensitivity detection was obtained. Additionally, targeted genes were amplified with single primer extension technology and a bead clean-up step was performed to discard unwanted fragments. The concentration optimization of libraries was performed using Qiaseq Quant Assay Kit (Qiagen), and all libraries were diluted to 4 nM. Libraries with different sample indices were combined in equimolar amounts in a final pool. As a next step, the final pool was sequenced in a Miseq System, (Illumina Inc., San Diego, CA, USA), according to manufacturer’s guide. The secondary analysis of fastq files was performed using Qiagen Clinical Insight-Analyse Universal with a panel-specific pipeline. Finally, the vcf files were clinically interpreted using Qiagen Clinical Insight-Interpret. Pathogenic, likely pathogenic variants and VUS in BRCA1, BRCA2, CDH1, PALB2, PTEN and TP53 genes were identified, reported and assessed in light of published evidence.

Results

Gene variants (pathogenic, likely pathogenic and VUS) in BRCA1, BRCA2, CDH1, PALB2, PTEN and TP53 genes were found in 53 (27.2%) of 195 patients. Of the cohort, 122 (62.6%) attended clinic with a positive family history. Furthermore, 121 (62.05%) were under 45 years of age and 48 (24.6%) met both family history and age criteria. In total, 58 mutations were identified with four patients having more than one mutation, while 14 novel previously unreported variants were detected. Of 53 patients, 20 had pathogenic variants, three had likely pathogenic variants and 35 had VUS.

Of the nine patient with BRCA1 variants, eight had different variants, three had the same mutation and one had two BRCA1 variants. Of the eight different BRCA1 variants, four were considered pathogenic and four were VUS.

There were 26 different BRCA2 variants identified in 32 patients, and of these 10 were pathogenic, one likely pathogenic and 15 were VUS.

Three different TP53 variants were detected in three patients, of which two were pathogenic and one was VUS. Eight different PALB2 variants were detected in eight patients, including two pathogenic, two likely pathogenic and four VUS. Five different CDH1 variants were identified in five patients, all of which were VUS. In this cohort, no variant was found in the PTEN gene. Detailed information about the patients (age of diagnosis, family history, gender), cancer stage, tumour characteristics [ER, PR, and human epidermal growth factor receptor 2 (HER2) status] and all information related with the detected variants (gene, location, nucleotide and amino acid change, exon number, impact, mutation classification, dbSNP number and HGMD variant class) can be seen in Tables 3, 4, 5, 6, 7 and Figures 1, 2, 3.

Discussion and Conclusion

BRCA1 is predominantly a breast cancer gene and for carriers the estimated lifetime for breast cancer ranges between 50–85% (10). In the present study, eight different BRCA1 variants were detected in nine patients, and four were considered pathogenic and four were VUS. Pathogenic variants were c.5266dupC (p.Q1756fs*74) and c.66dupA (p.E23fs*18) that are both frameshift mutations and c.1059G>A (p.W353*) and c.2800C>T (p.Q934*) that are both stop codon mutations. Frameshift mutations are particularly severe and cause changes in many bases downstream and can affect many of the amino acids in the protein. c.5266dupC (p.Q1756fs*74), which is one of the most common pathogenic variants in BRCA1 gene, was found in three patients (1.54%) in this study. According to the previous studies the estimated prevalence of the mutation is between 2.5% and 7.1%  in breast cancer patients (11). c.66dupA (p.E23fs*18), c.1059G>A (p.W353*) and c.2800C>T (p.Q934*) mutations have been reported previously, while the c.2800C>T (p.Q934*) mutation was previously recognized as a Japanese founder mutation (12). c.3833A>C (p.K1278T) and c.3082C>T (p.R1028C) mutations, which were both VUS and missense mutations, were found in the same patient. The c.3833A>C p.K1278T mutation has not been reported previously. In this case compound heterozygosity may be responsible for the disease. The c.1884T>G variant found in one patient, was not considered a disease-causing mutation in HGMD and was classified as VUS, and in ClinVar this variant was observed with hereditary breast and ovarian cancer syndrome (13). c.4358-3A>G, found in one patient in the present study, was a splicing mutation. The variant has no dbSNP ID yet and was previously identified in only two reports. One mentioned the susceptibility to breast and ovarian cancer and the other reported that it was observed associated with a cancer predisposition syndrome (14, 15). When we looked at the exons, exon 10 was remarkable. Out of the eight different mutations, five were in exon 10. In previous studies, similar data was given. Exon 10 was the most common location for BRCA1 mutations and mutations were predominantly distributed around exon 10 (16).

Germline mutations in the BRCA2 gene are highly penetrant for increased risks of breast and ovarian cancers, and male breast cancer, prostate cancer, pancreatic cancer, and melanoma (17). In this study 26 different BRCA2 variants were detected in 32 patients with operated breast cancer and 10 were considered pathogenic, one likely pathogenic and 15 were considered VUS. Pathogenic variants were c.8585dupT (p.E2863fs*6), c.1763_1766delATAA (p.N588fs*25), c.9027delT (p.H3010fs*18), c.5576_5579delTTAA (p.I1859fs*3), c.4471_4474delCTGA (p.L1491fs*12), c.4631dupA (p.N1544fs*4), c.8351G>A (p.R2784Q), c.5791C>T (p.Q1931*), c.7007G>A (p.R2336H) and c.1055dupA (p.Y352*). Most of the pathogenic variants were frameshift mutations (6/10) while frameshift insertion, stop codon, missense and a splicing mutation were identified. In the present study five patients had c.9976A>T p.K3326* variant which was considered VUS. The variant was a stop codon mutation and in HGMD it was reproted that the variant was a disease-associated polymorphism with additional supporting functional evidence. For these patients further studies are needed to identify the additional findings. Two patients had c.6427_6428delTCinsAT (p.S2143I) variant, which was considered a VUS. The variant had no dbSNP ID at the time of writing and was previously reported in only two studies. Both studies mentioned that the variant was observed with hereditary risk breast/ovarian cancer (18, 19). Besides this variant, there were 11 more variants in exon 11 found in our study. Exon 11 is very important for the BRCA2 gene, which comprises over 50% of the gene and encompasses half of the coding region. In our study, 12 mutations were found in exon 11 and four were pathogenic, one was likely pathogenic and seven were VUS. Out of four pathogenic variants, three were frameshift mutations and one was a stop codon mutation. We found c.4471_4474delCTGA (p.L1491fs*12) mutation in a male patient. This variant deletes four nucleotides in exon 11 of the gene and as a result, creates a frameshift and premature translation stop signal. Functional studies have not been reported for this variant but is expected to result in an absent or non-functional protein product (20). The c.4033G>T (p.D1345Y) variant, considered a VUS, has no dbSNP ID and has not been mentioned as a disease-causing mutation. In the literature one report was found that related this variant with rectal adenocarcinoma (21). The c.2608A>G (p.I870V) variant found was considered a VUS, was not been previously reported and had no dbSNP ID, like the other c.5206_5208delCAA (p.Q1736del) variant that was considered likely pathogenic. The novel BRCA2 mutation, c.5206_5208delCAA (p.Q1736del) was an in-frame 3 bp deletion that is predicted to result in the loss of one amino acid. A deletion is in-frame and if the reading frame of the gene is preserved and not disrupted, so a protein can be made and it may still be partially functional. These kinds of mutations are much rarer than the other types, such as stop, frameshift, and missense or splicing. In-frame deletions typically result in milder conditions but the functional impact and prognostic value of this novel in-frame deletion variant in BRCA2 remains to be elucidated. The c.6626T>C (p.I2209T) variant, considered a VUS, was not mentioned as a disease-causing mutation in HGMD but in ClinVar, it was associated with hereditary breast and ovarian cancer syndrome (22). The c.2926_2927delTCinsAT (p.S976I) variant has no dbSNP ID at present and was previously reported in only two reports. Both mentioned that the variant was observed in association with breast cancer (23, 24). Two missense variants in the BRCA2 gene, c.3503T>A (p.M1168K) and c.3310A>C (p.T1104P) and one variant in CDH1 c.2595G>C (p.W865C), were found in one patient, and all of them are considered as VUS. c.3503T>A variant was not mentioned as a disease-causing mutation. The c.8671A>G (p.T2891A) variant that we identified, is considered a VUS, but was not mentioned as a disease-causing mutation. However, one report was found and mentioned that it was observed with breast cancer (25). In another case we identified the c.353G>A (p.R118H) variant, previously reported once, and the case was suffering from different types of cancer, including not only breast, but also endometrial and ovarian cancer (26). The c.1055dupA, p.Y352* was another variant that was not reported previously. This duplication of one nucleotide creates a nonsense variant, which changes a tyrosine to a premature stop codon. It was predicted to cause loss of normal protein function and was considered pathogenic because of the protein truncation or nonsense-mediated mRNA decay (27). In another case, we identified the c.7481G>A (p.R2494Q) variant and according to the literature there are some conflicting findings related to pathogenicity (28).

Germline mutations in the TP53 gene cause a familial cancer predisposition, and carriers have a very high lifetime risk of malignancies, especially soft tissue sarcomas and breast cancer in women. The penetrance of breast cancer is very high and according to the National Cancer Institute Li-Fraumeni syndrome cohort cumulative incidence is 85% by age 60 years (29, 30). In the present study, three different variants were detected in three patients. All were missense mutation, one of which was a novel mutation, c.259C>T (p.P87S). The others had been reported previously and both were considered pathogenic.

Most recently, it was reported that 7% of all CDH1 mutations are present in non-gastric tumors, especially being identified in cases with breast cancer. In our study, five CDH1 gene variants were found, all of which were missense mutations and considered VUS. Four of them, c.1154C>G (p.P385R), c.2595G>C (p.W865C), c.1790C>G (p.P597R) and c.2560G>A (p.D854N) were all novel mutations and not previously reported. The patient with c.1154C>G variant also carried a pathogenic BRCA1 variant, c.66dupA (p.E23fs*18). Another case who had the c.2595G>C (p.W865C) variant also had two BRCA2 mutations, c.3503T>A (p.M1168K) and c.3310A>C (p.T1104P). The p.V475M variant (also known as c.1423G>A), results in a conservative amino acid change in the encoded protein sequence. This amino acid position is highly conserved. Although in silico tools predict a damaging effect on protein function, the data on variant occurrences in the general population are insufficient to allow any conclusion and the clinical significance of this alteration remains unclear (31, 32).

PALB2 mutations are found in 0.6 to 3.9% of families with a history of breast cancer. In previous studies, the estimated mean risk of breast cancer for female PALB2 mutation carriers by 70 years of age is 35% (33). In the present study, seven PALB2 gene variants were identified in eight cases, three of which were novel mutations and not reported before. The c.3285dupT (p.N1096*) mutation was considered likely pathogenic but has not been reported previously. The c.3388G>C (p.A1130P) and c.3447C>A (p.A1149A) variants were also previously unreported, were classified as VUS, and have no dbSNP ID. Another missense mutation, c.3428T>A p.L1143H, was identified in a patient who also had the BRCA2 pathogenic c.8351G>A (p.R2784Q) mutation. This variant in the PALB2 gene had no dbSNP ID, and was mentioned in only a few reports in the literature. In a study based in the Turkish population, this was observed with breast cancer (34). The c.194C>T (p.P65L) variant, considered a VUS, was not mentioned as a disease-causing mutation, but in the ClinVar database it was reported that this amino acid position was poorly conserved and thus the predicted effect will be tolerated by in silico analysis. Since supporting evidence is limited, the clinical significance of this alteration remains unclear (35). The c.833_834delTAinsAT p.L278H variant, classified as a VUS, and as functional studies have not been performed for this variant, the available evidence is insufficient to determine the role in the protein. BRCA1, BRCA2, TP53, CDH1, PALB2 and PTEN genes and lifetime risks for breast cancer are summarized in Table 6 (10).

Besides medical geneticists, breast surgeons and oncologists are well placed as a resource for patients who could benefit from genetic testing. The patients must be evaluated in the multidisciplinary councils and informed about the risks and benefits, and also discuss risk management strategies. The American Society of Clinical Oncology and the NCCN recommend considering genetic testing for breast cancer in patients, especially diagnosed at early age, with bilateral breast cancer, ER-/PR-/HER2- disease status, strong family history of breast and/or ovarian cancer, or a combination of these characteristics (9). The identification of an inherited pathogenic mutation predisposing to breast cancer in genetic testing does not mean that risk-reducing mastectomy is indicated. While risk-reducing mastectomy can be considered in BRCA1, BRCA2, PTEN and TP53, the situation can differ in other gene mutations. However, when combined with a significant family history of breast cancer, prophylactic surgery may be appropriate for patients with mutations in other genes (36) but the risks of the surgery should be discussed with the patient. In a multicenter study investigating the prevention of breast cancer with nipple-sparing mastectomy, prophylactic nipple-sparing mastectomy was performed in 346 patients who were carriers of BRCA1 and BRCA2. None of them developed breast cancer at the 56-month follow-up. In the risk analysis conducted in the same study, it was reported that if mastectomy was not performed, breast cancer was predicted to have developed in 22 of the patients (37). In another study, follow-up and prophylactic mastectomy were compared in order to reduce the risk of breast cancer in BRCA-positive patients. It was shown that mastectomy significantly reduced the risk of developing breast cancer, but had complications and risks (38). Prophylactic oophorectomy in premenopausal women with pathogenic variants in BRCA2 has also been shown to reduce the risk of breast cancer by approximately 50%. This rate is lower in BRCA1 patients who underwent risk-reducing oophorectomy (39). Advanced screening is recommended for patients with ATM, CDH1, CHEK2, NBN, NF1, PALB2, and STK11 mutations, but currently the data are insufficient to support risk-reducing mastectomy in the absence of other factors such as a strong family history. Risk is modulated by age, family history, and in some cases, a specific mutation in a particular gene. The guidelines broadly support the consideration of contrast and non-contrast breast magnetic resonance imaging and tomosynthesis mammography for annual screening, due to the increased risk of breast cancer in individuals with this group of mutations (36). As also seen in our study, studies assessing mutations, which are commonly identified in the young patient population, as treatment targets have been underway for a long time. Besides evaluating mutations in terms of recurrence, secondary malignancy development or familial risk, they have an important role in both treatment and prognosis, again emphasizing the importance of genetic analysis.

Analysis of the relationship between mutations and the histopathological characteristics of tumors show that BRCA1 mutations are associated with breast cancers that have a basal-like gene expression profile, high histological grade, negative estrogen receptor status, and HER2-negative (triple negative) status. BRCA2-associated breast cancers are usually high-grade, estrogen receptor-positive, and HER2-negative (40). The 5-year survival rate for patients with metastatic breast cancer is reported to be 27%, whereas for patients with metastatic triple-negative breast cancer associated with a BRCA1 mutation, this rate is only 11%. Therefore, new treatments that provide lasting benefits are needed in patients with advanced breast cancer associated with a germline BRCA mutation. Breast cancers with BRCA1 and BRCA2 mutations, are deficient in homologous recombination and disrupt the ability of cancer cells to repair DNA damage, and also known to be sensitive to both poly (ADP-ribose) polymerase (PARP) inhibitors and platinum agents (41). PARP is an enzyme family responsible for the cellular activities involved in DNA repair, such as the base excision repair pathway and genetic stability. PARP plays a key role in the repair of single-strand DNA breaks, which is important for the survival of the cell. The inhibition of PARP leads to continued single-strand DNA breaks, which causes replication forks to stop, and double-strand breaks to occur. In cells with normal homologous recombination, double-strand DNA breaks can be repaired by the homologous recombination repair pathway. However, in cells with homologous recombination deficiency, treatment with a PARP inhibitor results in cell cycle arrest, a concept called synthetic lethality, and apoptosis (42).

Tumors with changes that inactivate BRCA1/2, can respond to PARP inhibitors, such as olaparib, talazoparib, rucaparib or niraparib, and chemotherapeutics that cause DNA damage, such as cisplatin and carboplatin (43, 44). In the “triple negative trial”, a randomized phase III trial comparing carboplatin with docetaxel in patients with locally advanced metastatic or recurrent TNBC, patients with germline BRCA1/2 mutations showed significantly better response to carboplatin compared to docetaxel (41). Two randomized phase III trials reported PARP inhibitor efficacy compared to physician’s choice of chemotherapy in patients with metastatic breast cancer and one germline BRCA pathogenic variant. In the OlympiAD study (ClinicalTrials.gov descriptor: NCT02000622), olaparib was shown to improve progression-free survival with a hazard ratio of 0.58 [95% confidence interval (CI), 0.43–0.80] compared to standard chemotherapy. In the EMBRACA study (ClinicalTrials.gov descriptor: NCT01945775), talazoparib showed a progression-free survival contribution with a hazard ratio of 0.54 (95% CI, 0.42–0.71) (45). That these inhibitors offer oral treatments, and perhaps even more importantly, improve the quality of life of patients, makes them more attractive. In the NCCN Guidelines version 3.2022, both treatments are indicated as category 1 recommendation in the presence of a BRCA1/2 mutation. In addition to metastatic diseases, there are studies that also report the positive results of PARP inhibitors in neoadjuvant and adjuvant therapies (46).

CDH1 may also play a potential strategic role in the clinical management of breast cancer patients as a predictor of prognosis and survival. To date, CDH1 has not been defined as a molecular target for treatment, but in vitro studies have shown that the germ line function could be suitable for targeted therapy. Important new crosstalk mechanisms have been described. Growth factor signals are hyperactivated upon loss of CDH1, regardless of the somatic activating mutations in downstream effectors. In particular, the PI3K/Akt pathway is activated upon loss of CDH1 in the absence of specific oncogens. ROS1 gene rearrangements are a defined and approved therapeutic target in relation to different cancers. Bajrami et al. (47) have described a synthetic lethal interaction between CDH1 and ROS1. The authors showed that ROS1 inhibition in CDH1 defective breast tumor cell sequences and breast tumor xenografts derived from patients resulted in tumor cell death.

In contrast to the biomarker role of TP53, until recently very few studies were performed that target therapeutically mutant TP53. With the recent identification of several compounds capable of reactivating the mutant protein, however, PRIMA-1MET (p53 reactivation and induction of massive apoptosis, methylated derivative) also called APR-246 has been reported (48). In addition to inhibiting cell proliferation, APR-246 was seen to induce apoptosis and reduce migration in the investigated mutant p53 breast cancer cell sequences. Another anti-p53 compound, which has been investigated for anticancer activity in breast cancer cells is the 2-sulfo-pyrimidine molecule known as PK11007. Like APR-246, PK11007 stabilizes and reactivates mutant p53 (49). There are no FDA-approved therapeutic targets yet, either for CDH1 or T53, but ongoing studies are promising. Studies are ongoing to identify the other mutations identified in the present study, both to determine suitability as treatment targets and to define their characteristics predicting prognoses.

This study identified a total of 58 mutations in a cohort of 195 women who had been operated for breast cancer, of which 35 were VUS and 14 were novel variants that have not been previously reported. We have highlighted the VUS in this report because the clinical significance of VUS is changing over time and variant classification is important for clinical molecular genetic testing and clinical guidance. In a previous study, the researchers reclassified specific VUS identified over a 13-year period, and the results showed that 5.6% were reclassified as pathogenic or likely pathogenic variants. Further investigation into the pathogenicity of VUS is required. With continued monitoring of patients with VUS mutations, reclassification must be suggested when sufficient evidence is collected (50). Besides clinical follow-up, genetic follow-up should occur. Knowledge and identification of mutations have important implications for predictive, preventive, personalized medicine, besides genetic counseling and development of specific treatment protocols.

This study has also provided risk assessment data, which may be useful for clinical management for TP53, CDH1, PALB2 and PTEN, together with BRCA1 and BRCA2 genes in breast cancer patients and may also be important for surveillance of other family members. Further studies are needed to identify common variants in the Turkish population and to evaluate the pathogenity of VUS.

Ethics Committee Approval: The study was conducted in accordance with the Declaration of Helsinki and was approved by İzmir Katip Çelebi Non-Interventional Clinical Studies Institutional Review Board (0028/20.01.2022).

Informed Consent: Written informed consent forms were obtained from all patients.

Peer-review: Externally and internally peer-reviewed.

Authorship Contributions

Surgical and Medical Practices: A.S., Z.G.G., E.Ö.G., M.K.A.; Concept: A.S., Z.G.G., E.Ö.G., M.K.A.; Design: A.S., Z.G.G., E.Ö.G., M.K.A.; Data Collection or Processing: A.S., Z.G.G., E.Ö.G., M.A.T.; Analysis or Interpretation: A.S., Z.G.G., E.Ö.G., M.A.T.; Literature Search: A.S., Z.G.G., E.Ö.G.; Writing: A.S., Z.G.G., E.Ö.G.

Conflict of Interest: No conflict of interest was declared by the authors.

Financial Disclosure: The authors declared that this study received no financial support.