Diagnostics and treatment of ovarian cancer in the era of precision medicine - opportunities and challenges

Due to predictions of increasing incidences and deaths from ovarian cancer, this neoplasm is a challenge for modern health care. The advent of NGS technology has made it possible to understand the molecular characteristics of many cancers, including ovarian cancer. The data obtained in research became the basis for the development of molecularly targeted therapies thus leading to the entry of NGS analysis into the diagnostic process of oncological patients. This review presents targeted therapies currently in preclinical or clinical trials, whose promising results offer hope for their use in clinical practice in the future. As more therapeutic options emerge, it will be necessary to modify molecular diagnostic regimens to select the best treatment for a given patient. New biomarkers are needed to predict the success of planned therapy. An important aspect of public health is molecular testing in women with a familial predisposition to ovarian cancer enabling patients to be included in prevention programs. NGS technology, despite its high throughput, poses many challenges, from the quality of the diagnostic material used for testing to the interpretation of results and classification of sequence variants. The article highlights the role of molecular testing in ongoing research and also its role in the diagnostic and therapeutic process in the era of personalized medicine. The spread of genetic testing in high-risk groups, the introduction of more targeted therapies and also the possibility of agnostic therapies could significantly improve the health situation for many women worldwide.


Introduction
Ovarian cancer (OC) is diagnosed in a large number of women around the world annually -in 2020, 313,959 new cases were reported -significantly reducing their comfort level and also taking the lives of many of them (207,252 deaths) (1,2).OC ranks seventh in cancer incidence and is fifth leading cause of cancer-related death in women worldwide (1), in addition is considered to be the most lethal gynecological cancer (3).Statistical studies have shown a general downward trend in the incidence and mortality of ovarian cancer over the past decade linked, no doubt, to advances in medicine.However, the significant growth in incidence in younger women is worrisome (2).
It is known that the increased risk of developing ovarian cancer is related to genetic predisposition and family history (Hereditary Ovarian Cancer -HOC).Hereditary cancers, i.e. those caused by inherited mutations in genes that predispose to cancer development, account for as many as 20-25% of epithelial ovarian cancer cases (4).It is worth mentioning that breast and ovarian cancers show some common features at the molecular level (5).The distinct disease syndrome is Hereditary Breast and Ovarian Cancer (HBOC) in which the risk of breast and ovarian cancer is higher than in the general population (6).Breast cancer cases due to HBOC are estimated to account for 2 to 7%; for ovarian cancer, the range is 15 to 18% (4).
Whether spontaneously occurring or associated with a genetic predisposition, OC represents a major global problem and a challenge of modern medicine.Undoubtedly, the development of next-generation sequencing (NGS) technology has contributed to a better knowledge and understanding of cancer biology and, moreover, in the detection of high-risk groups (7).This is crucial in the early diagnosis of cancer and also in the selection of targeted therapy (8).NGS panels approved for use in patient diagnosis allow assessment of both somatic and germline variants, as well as single or global genomic changes (7).Such broad capabilities of NGS technology make it widely used in scientific research and patient diagnostics, including oncology, and an important step towards personalized medicine.However, many factors influence the success and improvement of NGS research, such as sequencing chemistry, sequencing technology, bioinformatics and data analysis (9).

Molecular pathogenesis of ovarian cancer
High-grade serous ovarian cancer (HGSOC) is the most common histological type of ovarian cancer and accounts for about 75% of ovarian cancers of epithelial origin (10).Despite the common histological features identified by pathologists, HGSOC tumors have significant differences in the clinical course of the disease, including response to treatment (11).HGSOCs exhibit significant heterogeneity due to differences at the molecular level (12).
The TP53 somatic mutation occurs as the first molecular alteration involved in HGSOC carcinogenesis and is referred to as a driver mutation.This condition was identified in 96% of HGSOC cases (3).The protein encoded by TP53, called p53, is a transcription factor that activates genes engaged in DNA repair, cell cycle and apoptosis after nonreversible DNA damage (13).Loss of p53 function promotes tumorigenesis, but does not itself cause a malignant phenotype, and at least one more genotoxic event is required.Most frequently, this is a BRCA1/2 mutation.BRCA1/2 can lose its functionality through somatic mutation, germline mutation or through promoter hypermethylation, which belongs to epigenetic modifications.It is worth noting that mutations are mutually exclusive with epigenetic silencing of the BRCA1/2 gene (3).BRCA1 and BRCA2 proteins are involved in DNA repair through homologous recombination (homologous recombination repair (HRR)) (14).Defects in HRR are estimated to occur in 50% of HGSOC cases.Besides changes in BRCA1/2, it can be caused by mutations in ATM, BRIP1, CHEK2, NBN, PALB2, RAD51B (3,14).However, an HGSOC tumor can acquire genomic instability through disruption in another pathway.BRCA1/2 loss of function is mutually exclusive with CCNE1 amplification and RB1 inactivation.Both CCNE1 and RB1 are involved in the Rb cell cycle regulation pathway (3).The protein cyclin E1 forms a complex with cyclin-dependent kinase 2 (CDK2) to promote cell cycle progression from G1 to S phase (15).Cyclin E1 expression relies on E2F transcription factors that are bound to retinoblastoma protein (Rb) (16).Amplification of CCNE1 and deletion of RB1 hasten the cell cycle, leading to defective S-phase progression and an elevated number of chromosome breaks, resulting in genomic instability.CCNE1 amplification appears in about 20% of HGSOC cases while RB1 inactivation occurs in 10% of HGSOC (3).

Targeted treatment optionsavailable now and under study
Currently, the standard of care for patients with stage II-IV ovarian cancer is platinum-based chemotherapy and surgical cytoreduction (16).Ovarian tumors with HRD are characterized by higher sensitivity to platinum and poly(ADP-ribose) polymerase inhibitor (PARPi) therapies.When RB1 function is lost, the tumor is also sensitive to platinum based therapies.In contrast, ovarian cancer with CCNE1 overexpression shows greater resistance to platinum compound therapies (17).Identifying the pathogenesis of the tumor at the molecular level allows the choice of targeted therapy.

PARP inhibitors monotherapy
For the treatment of breast and ovarian cancer, the Food and Drug Administration (FDA) has approved several PARPi.These drugs are effective for cancer patients with mutated BRCA1/2 and for treating recurrent platinum-sensitive ovarian cancer (18,19).A meta-analysis by Staropoli et al. of 1,839 epithelial ovarian cancer patients showed a statistically significant benefit from PARPi.A prolongation of survival time was confirmed, with no significant differences in progression-free survival (PFS) for the individual pharmaceuticals in this group (20).PARPi acts through synthetic lethality with loss of BRCA1/2 functionality.This occurs due to the destabilization of replication forks by PARPi, homologous recombination does not function properly in these cells and consequently DNA double-strand breaks (DSBs) occur, which cause cell death (21,22).Initially, PARPi was used only in patients with an identified BRCA1/2 mutation.However, the treatment is known to be effective for homologous recombination deficiency (HRD), which can be also caused by mutations in other genes.This approach has allowed a significant expansion of the group of patients in whom targeted therapy can be used (23).The use of PARPi in clinical practice has resulted in improved PFS.Despite its high efficacy, not all patients with tumors with mutations in BRCA1/2 or HRD respond to treatment, and moreover, most patients eventually develop resistance (24).

Combination therapies to improve PARPi efficacy and therapeutic strategies for PARPi resistance
Strategy that appears to be very promising in clinical practice is combination therapy involving PARP inhibitors and antiangiogenic drugs.The combination of these medications has been shown to result in a significant prolongation of PFS in both women with newly diagnosed advanced OC and with recurrent disease (25).Combinations with PI3K/AKT (phosphoinositide 3-kinase/protein kinase B), EGFR (epidermal growth factor receptor) and BET (bromodomain and extraterminal domain) inhibitors are proposed as other therapies that can overcome PARPi resistance (24).Noteworthy are the findings of Gasimla et al.The researchers analyzed the molecular profiles of BRCA2-deficient HGSOC tumors resistant to PARPi.They concluded that in these cases KRAS amplification is responsible for the resistance.The researchers suggest that inhibition of PLK1 (Polo-Like Kinase 1), which plays a role in regulating cell division, restores PARPi sensitivity (19).Immunogenicity plays an important role in the response to treatment.Kraya et al. showed differences in the immunogenicity of ovarian tumors with mutations in BRCA1/2 and distinguished two groups: tumors with PTEN loss and BRCA1 promoter hypermethylation, and tumors with wild-type PTEN.Cancers in the first group were characterized by significantly reduced CD3+, CD8+, and FOXP3+ T cell composition and higher HRD scores.The researchers suggest that combination therapy involving PARPi and immune checkpoint blockade (ICB) may be effective in patients with BRCA1/2 deficiency and wild-type PTEN (26).Shein et al. suggest that assessing the expression of immune-related genes may be an effective biomarker for estimating OS in women with ovarian cancer (27).The incorporation of epigenetic regulation into therapy was explored by Pulliam et al.Researchers have shown that inhibition of DNMT1 (an enzyme involved in DNA repair) sensitizes breast and ovarian cancers resistant to PARP inhibitor therapy.Moreover, DNMTi and PARPi therapy is effective regardless of BRCA status (18).Gupta et al. reported synergistic effects of histone deacetylase inhibitors (HDACi) and PARPi in vitro in ovarian cancer cells with an efficient homologous recombination mechanism.HDACi induces defective DNA repair, leading to an increase in the number of DSBs and, by repressing HR, enhances the action of PARPi.The researchers indicate that this therapy could have significant effects in poorprognosis tumors with CCNE1 amplification (28).The challenge is to develop a scheme to classify patients for monotherapy or combination therapy.Necessary for this is the search for biomarkers, especially at the molecular level (25,26).
Poly(ADP-ribose) glycohydrolase (PARG) inhibitors, similarly to PARPi, destabilize replication forks and induce DNA damage (21).However, PARG inhibitors show activity in BRCA wild-type cells, making them an effective alternative for patients with primary or acquired PARPi resistance (29).Successful completion of clinical trials on the efficacy of PARG inhibitors and a deeper understanding of their mechanism of action, as well as finding markers to predict susceptibility to therapy, is essential for the development of a new targeted drug effective in many types of cancer, not limited to ovarian cancer (21, 29).

Tumors with CCNE1 amplification
CCNE1 amplification is common in many types of cancer.Tumors with this molecular alteration are characterized by relative resistance to chemotherapy and, in addition, van Wagensveld et al. showed a lower content of immune cells which may result in a worse response of patients to immunotherapy (30).Therefore, finding targeted therapies for this genetic change has become a challenge for many researchers.Along with CCNE1 amplification, TP53 mutation is often present.In this group, significantly longer overall survival (OS) was observed in patients who received antiangiogenic therapy (31).One other strategy in cancers with CCNE1 amplification is CDK2 inhibition.The resulting CDK2-cyclin-E1 complex plays a role in chromosomal instability.Therefore, CDK2 inhibitors could inhibit tumorigenesis.CCNE1 amplification could be considered a biomarker of susceptibility to CDK2 inhibition.However, the oncogenic effect of cyclin E1 independent of CDK2 should be considered (32).Gallo et al. conducted studies in cell models and showed that CCNE1 overexpression results in susceptibility to PKMYT1 kinase inhibition.PKMYT1 kinase is a negative regulator of CDK1 and the selective inhibitor developed by the researchers appears to be an effective therapeutic strategy.The drug is intended for oral use, and clinical trials are currently ongoing for use as monotherapy or in combination with gemcitabine (33).High levels of cyclin E1 cause upregulation of the ATR axis, which induces the ATR/Chk1/WEE1 phosphorylation cascade (34).Xu et al. conducted studies on cell lines and xenografts with CCNE1 overexpression.The researchers showed that this genetic alteration causes sensitivity to WEE1 inhibition and ataxia telangiectasiaassociated Rad3 (ATR) inhibition (WEE1i-ATRi).Preclinical studies on a number of such drugs are currently underway and four ATRi and one WEE1i are in phase I -II clinical trials for efficacy in cancer therapy (35,36).Xu et al. suggest that by combining the two inhibitors, it will be possible to use lower doses of the drug, thereby reducing toxicity.However, the authors emphasize that treatment with WEE1i-ATRi, despite good tolerance in mice, requires in vivo studies before use in clinical practice (15).

Tumors with amplification of other genes
In addition to the most common amplification found in HGSOC which is CCNE1 amplification, somatic copy number alterations (SCNAs) also affect MYC, PIK3CA, KRAS and TERT and may also become targets for personalized therapy.Martins et al. show that MYC copy number correlates with the in vitro response to mTORC1/2 inhibition.Moreover, the researchers showed that activation of the mTOR survival pathway in ovarian tumors with MYC amplification is associated with a higher frequency of SCNAs in PI3K pathway genes.The coexistence of these two SCNAs also occurs in squamous cell lung cancer and triple-negative breast cancer (37).Meanwhile, Martins et al. demonstrated the efficacy of combination therapy involving mTOR pathway inhibition and paclitaxel in HGSOC tumors with MYC amplification (38).Transcription of the MYC oncogene depends on CDK7, CDK12, and CDK13, among others.Zeng et al. propose as a therapeutic strategy the use of THZ-1, which inhibits the aforementioned cyclin-dependent kinases which leads to a decrease in MYC expression levels and thus inhibition of tumor growth (39).

Tumors with RB1 inactivation
RB1 mutations are found in several cancer types including OC, which are further characterized by resistance to available therapies.Gong et al. tested 36 cell cycle inhibitors, of which the Aurora kinase inhibitors AURKA and AURKB showed the strongest synthetic lethality against small cell lung cancer (SCLC) cell lines.AURKA showed significantly higher selectivity compared to AURKB and was also well tolerated by mice (40).While Oser et al. demonstrated the efficacy of Aurora kinase B on SCLC cell lines with RB1 loss of function, they also point to good tolerance in experimental animals and suggest the possibility of using it in other types of cancer with this genetic alteration (41).
Figure 1 and Table 1 summarize the treatment options for women with HGSOC that may be possible in the near future.

Hereditary predisposition to ovarian cancer
Most cases of HOC and HBOC are determined by germline mutations in BRCA1 or BRCA2 (6,42).It is estimated that about 1 in 400-800 individuals carry the germline pathogenic variant of the BRCA1 and/or BRCA2 genes (43).Patients are placed under medical supervision if diagnosed as carrying a BRCA mutation.Chemoprevention or risk-reducing surgical treatment: riskreducing mastectomy and risk-reduction salpingo-oophorectomy Targeted treatment options for HGSOC approved by the FDA and under study described in the text.As for familial or hereditary ovarian cancer syndromes, they are caused by inherited mutations in tumor suppressor genes.In addition to BRCA1 and BRCA2 genes belonging to the Fanconi anemia pathway, mutations in PALB2, ATM, RAD51C/D and BRIP1 are also known to increase the risk of ovarian cancer.Another important pathway is the mismatched DNA repair pathway and genes such as MLH1, MSH2 and MSH6 (42).
Beyond the increased risk of developing breast and ovarian cancer, individuals in the HBOC group are at increased risk of developing other cancers, such as melanoma, pancreatic cancer and, in the case of men, prostate cancer (6).The risk of developing a specific cancer is related to the type of mutation.Women who have inherited the pathogenic BRCA1 variant tend to suffer from ovarian cancer at a younger age.In contrast, patients with a mutation in BRCA2 have a greater predisposition to develop male breast cancer, prostate cancer, pancreatic cancer and melanoma (45).Other HBOC-related genes include ATM, BRIP1, CDH1, PALB2, PTEN, RAD51C, and TP53 (46).Velaźquez et al. used a panel of 35 genes to study hereditary cancers.In the case of HBOC, pathogenic variants were noted in the following genes: BLM, BRCA2, BRIP1, CHEK2.The researchers underline that by using the expanded panel, they were able to detect pathogenic variants that would not have been detected with routinely used diagnostic panels (47).Resch et al. also demonstrated the benefits of using wider gene panels in their study.Most of the pathogenic variants identified were BRCA1, BRCA2.However, they also detected variants in CHEK2 and RAD51C and also in ATM, BARD1, MUTYCH and SMARCA4 which would not have been possible with a standard panel (48).
It should also be taken into account that the etiology of HBOC may be related to constitutional mosaic variants.These variants can involve somatic and also germinal cells, and can therefore be passed onto offspring.In their work, Hidalgo Mayoral et al. described a clinical case of a breast cancer patient in whom they detected a mosaic variant c.9648 + 1G>A in the BRCA2 gene.The authors point to other cases reported in the literature and emphasize the role of BRCA1/2 mosaic variants in HBOC.Due to the low allele frequency (VAF), many of these may have been missed in genetic analyses.A separate diagnostic algorithm is needed to detect potential mosaic variants using NGS sequencing (49).
Despite the significant genomic similarities between ovarian and breast tumors and the existence of the HBOC disease entity, these tumors show some distinguishing features.Weber-Lassalle et al. subjected samples of HBOC patients to genetic analysis.The researchers identified germline variants in BARD1 in breast cancer patients.Moreover, the presence of these variants was associated with an earlier onset of cancer (42.3 years) compared to the overall study group (48.6 years).BARD1 variants were not detected in ovarian cancer (50).

Molecular diagnostics of patients diagnosed with ovarian cancer
It is extremely important that testing is not limited to BRCA1/2.The benefit of expanding molecular analyses was demonstrated by Tao et al. 25% of HGSOC patients had genetic alterations other than disorders leading to BRCA loss of function.This gives patients a chance to receive off-label treatment or other therapies described in this review as part of their participation in clinical trials (51).
Currently available tests to assess HRD include: analysis of genes associated with homologous recombination repair, genomic "scars" and/or mutational signatures, and real-time functional assessment of HRD.It should also be remembered that the HRD phenotype is very complex and thus extremely difficult to assess reliably and unequivocally in a single analysis.These validated tests can be used to make treatment decisions for PARPi.However, they are not effective in predicting the group of patients who will not benefit from PARPi, i.e. they have no negative predictive value (23, 52).
Considering the mutually exclusive nature of the occurrence of BRCA mutations and CNNE1 amplification, the detection of highlevel amplification in combination with CCNE1 overexpression can be used as a criterion for predicting PARPi resistance (53).However, there are reports that genetic changes primarily thought to be mutually exclusive can co-occur.This is particularly true for loss of RB function, which appears to co-occur in tumors with BRCA1/2 mutations or other disorders of homologous pathway genes (54).Perhaps understanding this aspect will become possible through the use of spatial genomic, transcriptomic and proteomic technology.Spatial biology offers the possibility to analyze different cellular subclones and also complex interactions with the tumor microenvironment (TME), which together determine the process of carcinogenesis in an individual (55).
It should be noted that CCNE1 overexpression can occur without copy number amplification.Clinical trials have demonstrated the efficacy of WEE1i in both patients with CCNE1 amplification and overexpression.Therefore, future studies will probably evaluate the possibility of using CCNE1 expression instead of amplification of this gene as a marker of susceptibility to WEE1i therapy (56).
It is also worth mentioning that the use of PARPi prolonged PFS, but not OS.Therefore, it is important to consider the use of another therapeutic option.Unfortunately, immunotherapies in ovarian cancer have been less successful than in non-small cell lung cancer and melanoma.In order to improve the efficacy of this therapy, patient selection based on immunological profiling including assessment of high-grade microsatellite instability (MSIH), mismatch repair deficiencies (dMMR) and HRD is proposed (57).

Molecular tests for HBOC
In order for a patient to qualify for genetic testing, a woman must meet criteria outlined in national guidelines.These include age, hormone receptor status in BC, OC histology, and history of cancers that may be related to HBOC in the family.Although, these guidelines in different countries cover the same aspects however, the cutoff points may vary.There is a need to refine consistent recommendations between countries for monitoring mutation carriers specifically in genes other than BRCA1/2 (58).
The decision to use a multigene panel is up to the clinician.The physician, after analyzing the patient's phenotype and taking a health history, can choose the most appropriate molecular diagnostic option to identify HBOC, or to exclude it with higher probability.In addition to commercially available panel tests with a wide range, some laboratories also offer custom-designed panels after listening to the clinician's suggestions (45).
Despite improvements in diagnosis through the use of multigene panels, the issue of variants of uncertain significance (VUS) remains crucial.There is a need for algorithms as well as improved data flow and monitoring the situation globally to identify variants on which further research should be conducted to determine their clinical significance (59).These issues are discussed in more detail in the next section of the article.
Another important aspect is that HBOC tests should be tailored to different ethnic groups, as each has characteristic variants that occur with increased frequency compared to other groups.This approach can contribute to a better understanding of the molecular characteristics of specific communities of people and lead to the development of genetic tests dedicated to a specific community which will markedly improve the sensitivity and specificity of the test (60).

NGSopportunity, challenges, analysis report and interpretation of results
With the advent of sequencing technologies, we have seen their continued evolution.Sanger sequencing, referred to as firstgeneration sequencing, is limited to regions of interest within the genes of interest.The development of next-generation sequencing technologies has enabled comprehensive sequencing of larger regions of the genome.In the case of gene panels, gene fragments whose genetic variation is responsible for the occurrence of a defined phenotype are sequenced.It is also possible to sequence targeted protein-coding regions of all genes -whole exome sequencing (WES) and even whole genome sequencing (WGS), i.e. coding and non-coding sequences.NGS allows for more extensive analyses and is consequently more efficient due to the time and cost savings compared to Sanger sequencing (61).
Using advances in technology such as NGS, it is possible to make treatment decisions (62).Currently, biopsy material archived in the form of formalin-fixed paraffin-embedded (FFPE) blocks is used for molecular diagnosis of solid tumors (63).In this material, due to the fixation process, there may be changes in nucleic acids, their degradation and also modification of nitrogenous bases, which will be misread as a sequence variant in NGS analysis.To prevent these errors, researchers are developing new and more efficient protocols for nucleic acid isolation and library preparation (64).Another aspect of crucial importance in the fight against ovarian cancer worldwide is early detection and also monitor disease progression based on molecular testing.Many studies are looking into the possibility of using liquid biopsy, which has the undoubted advantage of being non-invasive (62).Analysis of cell-free tumor DNA (ctDNA) circulating in the blood, which can be performed using the NGS technique, has many potential applications such as early detection of both primary disease and recurrence, predicting prognosis, characterization of tumor heterogeneity and monitoring of treatment over time (7,65).However, the researchers point out that protocols for analyzing material obtained by liquid biopsy need to be refined, ctDNA enrichment methods need to be improved and the sensitivity of analytical techniques for detecting at low allele frequencies needs to be optimized.Further research is needed before liquid biopsy can be used in clinical practice (66).
The discovery of mutually exclusive pathways and also their interactions has become the basis for the development of new targeted therapies and, in the future, diagnostic regimens that qualify patients for treatment (67).Also in the case of approved drugs -PARPi, whose action is based on the concept of synthetic lethality, incoming data from studies show that it is essential to know the genetic context of the pathogenic change (68).For better insight and understanding of the clinical course of the disease, it is necessary to take into account not only individual genomic changes but their interrelationships, which together make up the characteristics of a given patient and thus determine the success of the planned therapeutic strategy (69).Consequently, there is a need to develop new biomarkers, the combined use of which will enable a better understanding of the patient's clinical condition and allow the selection of the optimal treatment pathway (68).
NGS sequencing has provided a wealth of data regarding the molecular biology of cancers, including HGSOC.However, it is now known that the use of bioinformatics analysis and tools is essential.It is through these that the pathways of carcinogenesis can be analyzed in depth (67).To detect homologous recombination deficiency, research uses methods based on whole-exome or whole-genome sequencing.However, in the practice of molecular diagnostic oncology, targeted sequencing panels are most often used.Therefore, the number of identifiable mutations is too low for HRD signature analysis, and as a result, many cases with HRD are not identified and patients do not receive PARPi treatment.Gulhan et al. propose a computational tool called Signature Multivariate Analysis (SigMA), which enables detection of mutation signatures from panel sequencing data.The researchers' impressive results indicate that the use of SigMA could significantly increase the number of patients who could be considered for targeted therapy (70).Schouten et al. developed a classifier that detects HRDassociated mutations based on evaluation of sequence variants in a panel of genes, epigenetic changes, and copy number alteration.The classifier proved to be an effective tool identifying most cancers with BRCA loss-of-function and BRCA-like phenotype, as well as all analyzed tumors with the RAD51C germline mutation (71).In contrast, Li et al. created a signature predictive of initial platinum resistance (IPR) not dependent on HRD but based on the differential expression of 11 genes.The researchers also identified significant differences in the characteristics of tumor infiltrating lymphocytes.The results suggest that patients with a high-IPR signature should show a better response to ICB (72).
In order for knowledge of the molecular profiles of tumors to be realistically reflected in clinical practice, that is, for treatment to be applied to a patient, it is important to keep in mind the significant heterogeneity of OC tumors, both temporally and spatially.For this reason, it seems reasonable that sequencing should also be performed after treatment and in relapse (73).This involves more research and thus cost generation.In silico data can be used to validate NGS assays instead of physical samples.In addition to high throughput and reliability, the use of in silico data makes it possible not to generate incremental costs associated with sequencing additional samples.These data can be successfully used to simulate hard-to-find gene variants, low-frequency variants or specific classes of variants (e.g., medium-sized insertions) (74).
The clinical course of the disease varies depending on the gene in which the pathogenic variant is present, whether the variant is germline, somatic, or an epigenetic modification, and the location of the change, i.e. which codon is affected (75).Therefore, correct and clear reporting and classify of detected changes is crucial.Somatic sequence variants are classified based on their clinical significance: tier I -variants of strong clinical significance; tier II -variants of potential clinical significance; tier III -variants of unknown clinical significance (VUS); and tier IV -variants of known non-significant significance (considered benign or probably benign).According to current guidelines, variants from tiers I-III must be included on the NGS analysis report, while it is not recommended to include lesions classified as tier IV.Tiers I and II have therapeutic, prognostic and diagnostic significance.VUS variants are the most challenging, and it is essential to report them which will enable researchers to decide later on their reclassification (76).Whereas germline variants are classified in a five-tier system: class 1non-pathogenic; class 2likely not pathogenic; class 3uncertain; class 4 -likely pathogenic; and class 5 -definitely pathogenic (77).Due to the distinctiveness of the scale used in the classification of somatic and germline sequential variants, it is helpful to include a verbal category name that indicates the pathogenicity of the variant to avoid potential confusion and misunderstanding.
Also extremely useful in the classification of variants are informatics tools that allow for the efficient flow of incoming data from NGS analyses.Hirotsu et al. demonstrated the utility of using MH BRCA and MH Guide developed by Molecular Health in determining the significance of VUS.The authors show significant value in confirming or exclusion of HBOC and also in predicting susceptibility to PARPi application when a variant of unknown clinical significance is detected (78).

Conclusions
Modern medicine has a great knowledge of the molecular-level changes involved in carcinogenesis, resulting in breakthroughs in patient diagnosis and treatment.Approaching cancer as if it were a disease determined by genetic changes and using NGS technology can significantly improve the prognosis of women with HGSOC.The use and utility of NGS testing in OC is summarized in Figure 2.However, despite its high efficiency, performance, NGS poses many challenges.Bioinformatics tools and the use of algorithms to obtain comprehensive and reliable molecular characterization of a patient's sample are helpful.It is crucial to properly interpret the test results and relate them to the patient's clinical condition.There is also a need to search for new therapeutic targets and conduct clinical trials of further targeted therapies.Before introducing new drugs into clinical practice, it is necessary to evaluate their efficacy, selectivity against tumor cells and also possible side effects.Clinical trials on the efficacy of new molecularly targeted drugs are being conducted around the world and Table 2 collects examples of such trials.A great opportunity may be the possibility of tumor-agnostic therapy, i.e. the use of targeted drugs regardless of the type of cancer just on the basis of finding the presence of a specific genetic change.Biomarkers should be sought to predict the response to the planned therapy.A very important aspect is the identification of women with a hereditary predisposition to ovarian cancer.As the range of therapeutic options grows, it is necessary to develop a Possibilities of using NGS technology in the and therapeutic process of ovarian cancer.laboratory diagnostic pathway that allows the best classification of patients for specific therapies.Pursuing these goals will contribute to more efficient diagnosis and treatment from an economic perspective and, invaluably, to improving the quality and prolonging the lives of patients.This can only be achieved through continuous improvement of skills and cooperation between clinicians and laboratory diagnosticians.

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TABLE 1
Targeted treatment options in OC available now and under study.

TABLE 2
Clinical trials of drugs that can be used to treat ovarian cancer.Multicenter Multiple Ascending Dose Study to Evaluate Safety, Tolerability and Pharmacokinetics of Quisinostat, a Histone Deacetylase Inhibitor, in Combination With Gemcitabine + Cisplatin Chemotherapy (Second Line for Patients With Non-small Cell Lung Cancer) or Paclitaxel + Carboplatin Chemotherapy (Second Line for Patients With Non-small-cell Lung Cancer, Second and Subsequent Lines for Patients With Epithelial Ovarian Cancer) Open-label Study of Safety and Efficacy of Quisinostat in Combination With Paclitaxel + Carboplatin Chemotherapy in Patients With Metastatic or Locally Advanced Epithelial Ovarian Cancer, Primarily Peritoneal or Fallopian Tube Carcinoma, Resistant to First Line Platinum and Paclitaxel Based Chemotherapy Ib Trial of the CDK4/6 Antagonist Ribociclib And The HDAC Inhibitor Belinostat In Patients With Metastatic Triple Negative Breast Cancer And Recurrent Ovarian Cancer With Response Prediction By Genomics (CHARGE) Trial of the ATR Inhibitor BAY 1895344 in Combination With Cisplatin and With Cisplatin Plus Gemcitabine in Advanced Solid Tumors With an Emphasis on Urothelial Carcinoma

TABLE 2 Continued
Phase 1b Study to Determine the Maximum Tolerated and/or Recommended Phase 2 Dose of the ATR Inhibitor Elimusertib (BAY 1895344) in Combination With PARP Inhibitor Niraparib, in Participants With Recurrent Advanced Solid Tumors and Ovarian Cancer Trial of Gemcitabine Combined With the Elimusertib (BAY 1895344) ATR Inhibitor With Expansion Cohorts in Advanced Pancreatic and Ovarian Cancer Study of M6620 (VX-970) in Combination With Gemcitabine Versus Gemcitabine Alone in Subjects With Platinum-Resistant Recurrent Ovarian or Primary Peritoneal Fallopian Tube Cancer Study of MLN8237, an Aurora A Kinase Inhibitor, Plus Weekly Paclitaxel or Weekly Paclitaxel Alone in Patients With Recurrent Epithelial Ovarian, Fallopian Tube, or Primary Peritoneal Cancer, Preceded by a Phase 1 Portion in Patients With Ovarian or Breast Cancer Study of MLN8237, a Novel Aurora A Kinase Inhibitor, in the Treatment of Patients With Platinum-Refractory or Platinum-Resistant Epithelial Ovarian, Fallopian Tube, or Primary Peritoneal Carcinoma Drug Interaction Study of JI-101 & Everolimus in Advanced Solid Tumors, Expansion Pharmacodynamic Study of JI-101 in Advanced Low Grade Endocrine Tumors, Ovarian Cancers or K-RAS Mutant Colon Cancers Study of the Oral PARP Inhibitor Olaparib With the Oral mTORC1/2 Inhibitor AZD2014 or the Oral AKT Inhibitor AZD5363 for Recurrent Endometrial, Triple Negative Breast, and Ovarian, Primary Peritoneal, or Fallopian Tube Cancer Clinical Study to Evaluate Dual mTORC1/2 Inhibitor (ATG 008) or Selective Inhibitor of Nuclear Export Compound (ATG-010) in Combination With Chemotherapy in Patients With Relapsed or Metastatic Ovarian Cancer, Endometrial Cancer, and NCT04726332 A Dose Escalation and Expansion Study of the Safety and Pharmacokinetics of XL102 as Single-Agent and Combination Therapy in Subjects With Inoperable Locally Advanced or Metastatic Solid Tumors Study of SY-1365, a Selective CDK7 Inhibitor, in Adult Patients With Advanced Solid Tumors Phase 1 Data from ClinicalTrials.govavailable as of July 31, 2023.