Importance of hereditary variants in ovarian cancer and endometrial cancer

1. Introduction

Hereditary variants play a significant role in all cancer types and are responsible for approximately 10% of diagnosed cancer cases [1]. Detection of these variants has vital importance in affected patients as identifying cancer-related variants helps with prognosis, follow-up, systematic treatment and surgery steps [2]. Many predisposing genes have been reported related to cancer types, most of which are tumour-suppressing and demonstrate an autosomal dominant inheritance [3]. For hereditary breast and ovarian cancer (HBOC), BRCA1 Dna Repair-Associated Protein—BRCA1 and BRCA2 Dna Repair-Associated Protein—BRCA2 are the best-known predisposing genes [3].

The DNA repair genes (BRCA1 DNA Repair-Associated Protein—BRCA1, BRCA2 Dna Repair-Associated Protein—BRCA2, BRCA1-Interacting Protein 1—BRIP1, RAD51 Paralog D—RAD51D, RAD51 Paralog D—RAD51C, Partner and Localizer Of BRCA2—PALB2, ATM Serine/Threonine Kinase—ATM, ATR Serine/Threonine Kinase—ATR and APC Regulator Of Wnt Signaling Pathway—APC) and mismatch repair genes (DNA Mismatch Repair Protein—MLH1, MutS Homolog 2—MSH2, MutS HOMOLOG 6—MSH6 and PMS1 Homolog 2, Mismatch Repair System Component—PMS2) are associated with cancer types. Mutations that occur especially in the MLH1 and MSH2 genes directly affect the protein dimer structure and cause loss of function in the obligatory and secondary partners of protein [4]. These genes’ pathogenic variants play an important role in diagnosing ovarian and endometrial cancer [5].

Ovarian cancer (OC) is the eighth most-common cancer in terms of mortality in women [6]. For treatment in recent years, poly-ADP-ribose-polymerase (PARP) inhibitors have been used. Treatment is defined according to molecular genetic analysis and the variants detected in the BRCA1 and BRCA2 genes related to the homologous recombinant repair (HRR) genes [7]. The appearance of a deficiency in these genes increases the sensitivity to PARP inhibitors [8].

Endometrial cancer (EC) is rarer than OC and is detected mainly in the postmenopausal phase; in recent years, however, the incidence of EC has been rapidly increasing [9]. Endometrial cancer has subtypes that are classified according to histopathological results. Even if these results have a powerful effect on prognosis, molecular genetics results are required [10].

This study aimed to illuminate the critical role of hereditary variants in the development of OC and EC. Recent advancements in next-generation sequencing technology have significantly streamlined the identification of these genetic variants, thereby making the process not only more accessible but also cost-effective. In the contemporary landscape of cancer diagnostics, multigene-targeted cancer panels have emerged as essential tools that provide comprehensive insights that facilitate more accurate diagnoses and personalised treatment strategies. Notably, this research introduces novel perspectives on the interplay between hereditary genetics and cancer susceptibility, thereby contributing to a deeper understanding of these complex diseases.

By using next-generation sequencing technology the detection of these variants became more easily at a low cost. Multigene-targeted cancer panels are very important nowadays for the cancer diagnosis process.

2. Methods

2.3 Next-generation sequencing (NGS)

In this study, we performed a targeted multigenic cancer panel. NGS technologies include several key steps, which are the fragmentation of genomic DNA, adding an adapter ligation for indexing and targeted panels, amplification of targeted genes or preparation of library and sequencing part. Sequencing depth and coverage are the main points of NGS data analysis, here in our targeted cancer panels we use 20× coverage for reading. The quality control of the NGS data it is dependent on a quality Q score of 30 (Q30) corresponds to a 99.9% accuracy rate, which is typically considered acceptable. By meticulously addressing these aspects of library preparation, sequencing depth, and quality control metrics, researchers can enhance the reproducibility and transparency of their NGS studies.

Cancer predisposition genes were analyzed with a NextSeq-550-Illumina NGS system (Illumina, San Diego, CA, USA) and Qiaseq Targeted (Qiagen) DNA panel (3891121889, Qiagen, Hilden, NW, Germany). In the Trusight Illumina NGS system, 94 predisposition cancer genes were included shown in Fig. 1, and in the Qiaseq Targeted DNA panel 93 genes were shown in Fig. 2.

Fig. 1.Trusight Illumina Next-Generation Sequencing system 94 predisposition cancer genes.

Fig. 2.QIAGEN Targeted DNA Panel Next-Generation Sequencing system 94 predisposition cancer genes.

NGS technology has three essential steps: library preparation, sequencing and data analysis.

3. Results

In our study, we analysed 50 patients with OC and 20 patients with EC. The identified variants played a vital role in diagnosis and genetic counselling. Of the 50 OC cases, we detected variants in 37 cases. In total, 46 variants were detected, 11 (23.9%) of which were pathogenic/likely pathogenic, 1 was novel and 35 (76.1%) were classified as VUS as shown in Tables 1 and 2. Thus, the diagnostic rate for OC in our study was 18%.

BRCA1 variants are the most frequently found variants in OC. In our study, we detected three pathogenic/likely pathogenic (P/LP) variants and one VUS variant in the BRCA1 gene. We also detected pathogenic variants in the Menin 1—MEN1, Exocyst Complex Component 1—EXOC1, Rad51 Recombinase—RAD51, Atm Serine/Threonine Kinase—ATM, Nibrin—NBN, Activin A Receptor, Type Ib—ACVR1B, Tumor Protein P53—TP53 and Dna Mismatch Repair Protein Mlh1—MLH1 genes.

Using a targeted gene panel for the 20 EC cases, we detected variants in 12 cases. In total, we detected 17 variants, 6 (35.2%) of which were pathogenic/likely pathogenic and 11 (64.7%) were VUS variants, as shown in Tables 3 and 4. For EC, the diagnostic rate was 30%.

We also detected P/LP variants in the ATM, MLH1, MSH2 and BRCA2 genes in the EC cases. In both of the cancers, the number of the VUS variants detected was higher than that of the P/LP variants. The VUS variants must be followed up every six months and the cases must be informed.

In addition, we detected a benign TP53 NM_000546.5:c.215C>G (p.Pro72Arg) variant in 25 OC cases and 9 EC cases.

Functional analysis plays a crucial role in the interpretation of genetic variants, particularly those classified as variants of uncertain significance (VUS). Functional analysis provides a systematic approach to evaluating how specific genetic alterations influence protein function, cellular pathways and overall organismal behavior. By elucidating the effects of VUS, researchers can distinguish between benign variants and those with pathogenic potential, thereby enhancing the reliability of genetic diagnoses. Unfortunately, we could not perform functional analysis in our laboratory.

4. Discussion

Ovarian and endometrial malignancies result in considerable diagnostic, therapeutic and management issues [11, 12]. Inherited variants play a critical role as they have a substantial impact on an individual’s susceptibility to various diseases, including cancer. These variants can greatly raise the chances of getting cancer [13].

The DNA mismatch repair (MMR) pathway is a highly conserved mechanism that is crucial for maintaining genome stability. Defects in MMR are associated with genome-wide instability and increased susceptibility to specific cancers [14]. Detecting cases with these genetic risks means that special tests can be performed and preventive steps, such as more frequent check-ups or surgeries, can reduce the risk of cancer or detect it early, when it is easier to treat.

Here we present the germline genetic mutations observed in ovarian cancer patients that were shown in our Medical Genetics department, as determined through NGS analysis. NGS technology is widely used for disease diagnosis, prognosis, therapeutic decision-making and follow-up of patients and is becoming more important for the detection of new variations and mutations [15]. The liquid biopsy process is one of the new variant detection methods that is an easy step, especially when the investigation needs to be performed from biopsies [16].

In addition, DNA repair genes are important in cancer cases; in our study, we detected the likely pathogenic variant NM_000249.4(MLH1):c.1690C>T in one of the OC cases (OV-8). The same case had a pathogenic NM_007294.4(BRCA1):c.5266dupC variant (see Table 1). The BRCA1:c.5266dupC variant has been reported as a founder effect and is the most common variation among patients diagnosed with breast cancer and OC, with a frequency of 5.47% [17].

In a molecular genetic analysis of 1197 individuals with a familial history of breast cancer and/or OC in Greece, Romania and Turkey, a pathogenic variant was identified in 264 (22.1%) cases, while a VUS was identified in 34.8% of the cases. A significant 43.6% of the detected variants were located in the BRCA1/2 genes [1]. Another study detected the highest number of pathogenic somatic mutations in high-grade serous carcinoma (HGSC) patients in the TP53 and BRCA1 genes in 86 OC cases [18]. Santonocito et al. [19] presented a spectrum of detected BRCA1/2 in 517 of 2351 variants in Italian breast cancer and/or OC cases.

We detected a pathogenic novel variant in OV-6 case, a nonsense NM_020327(ACVR1B):c.979C>T variant that is related to pancreatic cancer. Our patient was diagnosed with ovarian cancer, in recently published literature this gene was investigated in various cows with cystic ovarian disease and the results were significant [20]. ACVR1B gene has also a main role in ovarian follicular development [21]. ACVR1B gene plays a crucial role in the signaling pathway mediated by Transforming growth factor-beta (TGF-β) family members, such as activins and inhibins [22] but the certain role in ovarian cancer it is not yet revealed. The gene function or detected variants in ACVR1B need to be investigated in functional analysis.

In EC cases, we detected three variants related to DNA repair genes: an NM_000249.3(MSH1):c.790+1G>A variant in case E1, a missense NM_000251.2(MSH2):c.274C>G variant in case E4 and a frameshift variant in case E5 (see Table 3).

The tumor-suppressing gene TP53 stands out as the most commonly mutated gene across various human tumors, thus implicating its pivotal role in tumorigenesis [14]. We detected a missense NM_000546.6(TP53):c.493C>T variant in the OV-7 case in our study. The ATM gene also has an important role in the homologous recombination (HR) repair process; however, ATM signalling remains relatively limited in comparison to other DNA damage response (DDR) molecules, such as PARP and ATR, but ATM’s intricacies hold promise for advancing targeted therapeutic strategies in cancer treatment [23]. Ongoing clinical studies are investigating the efficacy of PARP inhibitors, platinum combinations, ATR inhibitors and immunotherapy in patients with germline BRCA mutations [24].

Although multiple assays have been developed to forecast homologous recombination deficiency (HRD), determining the most reliable predictor of drug response remains elusive. There have been nine FDA PARPi approvals/indications in OC since 2014 [25].

The results of forthcoming clinical trials are eagerly anticipated to provide clarity and guide treatment approaches in this rapidly evolving domain [16]. Certain PARP inhibitors, including niraparib and rucaparib, have been licensed by the FDA for the treatment of advanced or recurrent EC with particular molecular features, such as HRD [15]. PARP inhibitors offer significant clinical benefits for patients with BRCA1/2 mutations (BRCAm) [16].

In the context of genetic counseling, the management of variants of uncertain significance (VUS) presents unique challenges that require a nuanced approach. Clinicians should emphasize that these variants are neither definitively pathogenic nor benign. Clear communication is essential to help patients understand the potential implications for their health and the significance of uncertainty in genetic testing results. Clinicians should conduct thorough risk assessments based on the patient’s personal and family medical history and should consider collaborating with genetic counselors or specialists who can provide additional expertise in interpreting VUS. Periodically they must reevaluating VUS in light of new evidence, clinical guidelines and updated databases. This may involve reinterpreting variants as additional data become available, potentially leading to reclassification that could impact patient management. By integrating these strategies into clinical practice, clinicians can effectively manage VUS, enhancing patient understanding and care while navigating the complexities of genetic information.

We acknowledge the importance of experimental validation in confirming these functional implications. The follow-up studies that need to be performed involve various functional assays to assess the impact of the identified VUS on relevant biological processes. These experiments will include cellular assays to evaluate changes in cellular behavior, as well as biochemical assays to investigate alterations in protein function. We believe that this experimental validation will not only strengthen our findings but also provide invaluable insights into the pathogenicity of these variants.

Overall these findings of the detected variants give information about the disease risk assessment, inherited variant predispositions can be used in prevention strategies, targeted screening procedures and early intervention in some cases. One another important part is precision medicine according to the variation results the treatment methods are planned. Inherited genetic variants provide valuable insights into family health history and they are important in genetic counseling.

5. Conclusions

Inherited variants play a pivotal role in the development and progression of ovarian and endometrial cancers. Their influence on disease susceptibility underscores the importance of genetic screening and risk assessment, enabling targeted interventions for at-risk individuals. Early detection and personalized management strategies, informed by an understanding of inherited variants, offer promise for improving outcomes and reducing the burden of these gynecological malignancies. This study has several limitations that warrant consideration. Firstly, our analysis was restricted to the genes included in the targeted panel, which may not encompass all potential genetic contributors to ovarian and endometrial cancers. As cancer is a complex disease influenced by a multitude of genetic factors, it is plausible that variants in other genes, not covered by our panel, could also play a significant role in the etiology of these cancers. To gain a more comprehensive understanding of the genetic landscape associated with these diseases, whole exome sequencing (WES) or whole genome sequencing (WGS) could be beneficial. These approaches would allow for the identification of a broader range of variants, including those in non-coding regions, and may uncover additional genetic alterations that contribute to cancer development. Furthermore, in the context of cancer, the assessment of microsatellite instability (MSI) is crucial, as it is associated with the effectiveness of certain therapies and provides insights into tumorigenesis. Additionally, both ovarian and endometrial cancers are considered rare in our region when compared to other cancer types. The limited number of cases may restrict the diversity of the variant spectrum observed in our study, potentially hindering our ability to draw robust conclusions about their clinical implications. If the incidence of these cancers were higher, it is likely that we would encounter a more varied set of genetic variants, which could provide more specific and actionable insights in terms of diagnosis and treatment strategies. Addressing these limitations in future studies could significantly enhance our understanding of the genetic factors associated with ovarian and endometrial cancers.

Availability of data and materials

The data supporting the findings of this study are available with the corresponding author upon request.

Author contributions

DZ, HG—Concept and design. SY, DZ, HSG—Resources. SY, DZ, HSG, EIA, EA—Materials. SY, DZ, HSG, EA, SD, HG—Data collection and/or processing. DZ, HSG, EA, SD, EIA, EA —Analysis and interpretation. DZ, HSG—Literature review. DZ, HSG, SY, HG—Writing manuscript. SY, HG—Critical review.

Ethics approval and consent to participate

This study was approved by the Ethics Committee of Trakya University (2023/71). All patients agreed to participate in this study.

Acknowledgment

Not applicable.

Funding

This research received no external funding.

Conflict of interest

The authors declare no conflict of interest.