**2.3 Molecular pathology of ovarian high-grade serous carcinomas**

Massive parallel sequencing studies have revealed that ovarian HGSCs are characterised by somatic *TP53* mutations more commonly in the DNA binding domain in high frequency (>95%) [36, 37]. They have also demonstrated genomic alterations in the homologous recombination repair (HRR) pathway leading to genomic instability and aneuploidy characterised by high copy number structural alterations (CNAs). CNAs can be recognised as oncogene amplifications such as *CCNE1* (20%), *MECOM, EMSY* and *MYC* and deletions/breaks of tumour suppressor genes such as *PTEN, RB1, RAD51B* and *NF1* [38, 39]. Additionally, recurrent mutations have been observed in a variety of genes such as *NF1* (4%–6%), *RB1* (2%–6%)and *PTEN* (<1%) along with structural alterations/deletions can result in genes inactivation in relatively high frequency such as 20%, 17% and 7%, respectively [38, 40]. Ovarian HGSCs display genomic alterations in *BRCA1/2* genes that are involved in the homologous recombination repair (HRR) pathway. Almost 15% of HGSCs have *BRCA1/2* germline mutations, 5% somatic mutations and 11% show *BRCA1* promoter epigenetic silencing through CpG islands hypermethylation [38, 41]. Mutational alterations have also been observed in other HRR-related genes resulting in an HRR deficient phenotype in 50% of the cases and leading to high genomic instability [42]. These HRR-related genes include Fanconi anaemia genes (*PALB2, FANCA, FANC1, FANCL, FANCC), RAD* family genes (*RAD50, RAD51, RAD51B, RAD51C, RAD54L),* MRN complex genes [*Mre11-Rad50-Nbs1(Nibrin)]* and DNA damage response (DDR) genes (*ATM, ATR, CHEK1, CHEK2)* [42, 43]. Based on the HRR pathway status, HGSCs can be categorised into two morphologically distinct histotypes. *HRR- proficient tumours* demonstrate papillary, micropapillary and slit-like space architectural patterns with worse prognosis, while *HRR-deficient (HRD*) *tumours* show SET-like morphology (solid, endometrioid and transitional patterns) and improved progression-free survival due to beneficial responsiveness to platinum and poly ADP-ribose polymerase (PARP) inhibitors [26, 44]. HRR-proficient tumours are more likely to be resistant to these therapeutic interventions and are characterised by genomic alterations unrelated and mutually exclusive to *BRCA1/2* pathway, such as *CCNE1* gene amplification [45, 46].

According to NCCN guidelines HRD status should be tested in order to optimise the PARP inhibitor HGSCs treatment. Most of the HRD assays evaluate the status of germline or somatic HRR gene mutations and the presence of genomic instability by analysing the percentage of genomic loss of heterozygosity, telomeric allelic imbalance and genome-wide structural alterations (HRD mutation signatures) [47–49]. It should be noted that primary resistance to PARP inhibition can be observed in HGSCs with functional HRR, particularly in the presence of CCNE1 amplification. Additionally acquired resistance or decreased sensitivity to PARPi therapy can occur through HRR genes functional restoration by secondary mutations [50, 51]. In this setting, a functional assay can be used by evaluating the HRD status at RNA or protein levels [52, 53].

Other prognostic and treatment predictive biomarkers in HGSCs include tumour molecular subtyping based on transcriptional profiling that divides HGSCs into four categories (differentiated, immunoreactive, mesenchymal and proliferative), the former (differentiated and immunoreactive) with favourable biological behaviour

and prognosis and the latter (mesenchymal and proliferative) with aggressive clinical course and worse prognosis [54–56]. In addition, promoter hypermethylation of *TAP1* gene in 6p21.3 chromosomal locus confers an unfavourable prognosis, while an increased count of CD8+ tumour infiltrating T lymphocytes is associated with favourable outcome [57–59]. Protein expression studies on PD-L1, LAG3 and potential use of immunotherapeutic modalities on ovarian HGSCs have demonstrated modest therapeutic results and controversial prognosis [60–62]. Other dysregulated pathways in HGSCs are the *PIK3CA/AKT* and *NOTCH* pathways which can be therapeutically targeted by using PIK3CA or AKT inhibitors [63, 64], whereas *HER2* overexpression/ amplification (2–4% in HGSCs) has no significant impact on prognosis, albeit a finding that can be exploited for anti-HER2 targeted therapy [65, 66].
