Recent Advances in Classification and Histopathological Diagnosis of Ovarian Epithelial Malignant Tumours

Abstract

Ovarian tumours are a heterogeneous group of neoplasms classified based on histopathologic type and grade of differentiation. They comprise a broad range of tumours from benign and borderline to malignant histotypes characterised by different histopathological, immunophenotypic and molecular features. The purpose of this chapter is to present an overview of the recent advances in the ovarian epithelial malignant tumours classification along with the histopathological, immunophenotypic and molecular diagnostic criteria highlighting areas of terminology discrepancies or changes and diagnostic challenges. These changes provide a better understanding of the ovarian tumours nature and lead to a more efficient therapeutic management of these pathological entities.

1. Introduction

Ovarian cancer is the most lethal malignancy of gynaecological cancer representing 23% of gynaecological neoplasms and the 5th most common leading cause of death in women. Most ovarian malignant neoplasms are diagnosed at an advanced stage with high recurrence rates and an overall 5-year survival rate of around 50% [1, 2, 3].

Ovarian tumours originate from ovarian or fallopian tube tissue and are divided into epithelial tumours (benign, border-line and malignant), germ cell tumours, sex cord-stromal tumours and mesenchymal tumours [4].

In this chapter, we will focus on the epithelial ovarian malignant tumours and will present an overview of the recent advances in the ovarian tumours classification along with their histopathological, immunophenotypic and molecular features.

Epithelial ovarian malignant tumours comprise a heterogeneous neoplastic disease with distinct histomorphologic features, pathogenesis, precursor lesions, immunophenotypic and molecular profiles, different biological behaviour and clinical outcomes [4]. According to the recent 2020 WHO classification based on histomorphology, immunophenotypic features and molecular alterations, epithelial ovarian malignant tumours are classified into five main types with different incidences: high-grade serous carcinoma (HGSC—70%), low-grade serous carcinoma (LGSC—5%), endometrioid (EC—10%), mucinous (MC—3%) and clear cell carcinomas (CCC—10%) [3, 4, 5, 6].

Rare histopathologic entities are seromucinous carcinoma, malignant Brenner tumour, mesonephric-like carcinoma, undifferentiated and dedifferentiated carcinomas, carcinosarcoma and mixed cell carcinoma [5]. Seromucinous carcinoma as a distinct entity, characterised by serous and endocervical-type mucinous epithelium with foci of clear cells and areas of endometrioid and squamous differentiation, has been removed from the recent 2020 WHO classification. It is now considered a subtype of endometrioid carcinoma based on immunohistochemical and molecular studies [5].

Traditional concepts of ovarian carcinogenesis assumed that ovarian cancer pathogenesis is due to Müllerian or non-Müllerian metaplasia of ovarian surface epithelium leading progressively to malignant transformation [7] or due to malignant transformation of endometriosis lesions and/or inclusion cysts [8]. Recent pathological observations and molecular studies have revealed that the majority of the high-grade serous carcinomas arise from a precursor dysplastic lesion in the fimbria of fallopian tubes, designated as STIC (Serous Tubal Intraepithelial Carcinoma), whereas low-grade serous carcinomas arise within the ovarian parenchyma from benign or borderline serous tumours [9, 10, 11]. In addition, the presence of genomic alterations in the BRCA1 and BRCA2 tumour suppressor genes and gene mutations in p53, p16, CCNE1, BRD4 and RSF1, and centrosome copy number abnormalities, in STIC and high-grade serous carcinoma, suggest a clonal histogenetic relationship between this precursor lesion and HGSC [12, 13, 14, 15]. A clonal histogenetic relationship has also been observed among endometriosis, precursor ovarian surface epithelium lesions and endometrioid and clear cell carcinomas based on mutations found in ARID1A, PIK3CA, KRAS and MET genes among these pathologic entities [16, 17, 18].

It is now well established that ovarian carcinogenesis is based on a dualistic model of pathogenesis that divides ovarian epithelial malignant tumours into two main categories, designated as type I and type II ovarian tumours [7].

Type I ovarian epithelial tumours arise from precursor lesions in the ovary such as cystadenoma or adenofibroma. These lesions can undergo malignant transformation through atypical proliferation or transformation to borderline tumours and eventually into invasive ovarian neoplasms. Type I ovarian epithelial tumours include low-grade serous carcinoma, endometrioid carcinoma, mucinous carcinoma, clear cell carcinoma and malignant Brenner tumour. They have a relatively indolent clinical course and are characterised by genomic stability, distinctive molecular profile for each histotype and low incidence of p53 mutations (10–13%), except of mucinous carcinoma, which displays high incidence of p53 mutations (64%) [19, 20].

Type II ovarian epithelial tumours arise from distal fallopian tube fimbria epithelium through dysplastic lesions (STIC) and finally towards invasive carcinomas. They show aggressive biological behaviour and comprise high-grade serous carcinoma, carcinosarcoma and dedifferentiated and undifferentiated carcinomas [6, 21]. They are characterised by genomic instability, high incidence of p53 mutations and abnormal function of tumour suppressor genes BRCA1 and BRCA2 due to mutations or gene promoter hypermethylation [19, 20].

In the following sections, we are going to present the morphologic, immunophenotypic and molecular alterations that can be found in the five main histotypes of ovarian epithelial malignant tumours and the putative implications that may have in the clinical outcomes and targeted therapeutic interventions.

2. High-grade serous ovarian carcinomas (HGSCs)

High-grade serous carcinomas (HGSCs) account for 70% of ovarian carcinomas and represent the most aggressive and chemoresistant epithelial ovarian neoplasms. Most patients are postmenopausal women and admitted to the hospital at advanced clinical stage (80%) with extra-ovarian metastasis and thus with a high incidence of mortality around 70–80% globally [22].

Common predisposition factors are infertility, menopausal hormonal therapy and the hereditary breast and ovarian cancer (HBOC) syndrome characterised mostly by germline BRCA1/2 genomic alterations [23] and less frequently (2%) by germline genomic alterations in other homologous recombination repair (HRR) genes such as ATM, BRIP1, RAD51C and RAD51D [24, 25].

2.1 Pathology of ovarian high-grade serous carcinomas

These tumours are large, exophytic and usually bilateral with solid and papillary growth patterns and fluid-containing cysts. Necrosis is not uncommon and extraovarian affected sites can be observed. Occasionally a small tumour nodule can be found at the distal part of the fallopian tube fimbria.

Microscopically, HGSCs are heterogeneous tumours displaying solid, papillary, glandular and/or cribriform architectural patterns, sometimes with slit-like spaces. Recently, two histotypes have been observed, the classic type and the SET type (solid pseudoendometrioid and transitional variant) [5]. Classic type HGSCs exhibit papillary, micropapillary and solid growth patterns. The neoplastic cells demonstrate large pleomorphic nuclei with prominent nucleoli, high mitotic activity (>5 mitoses/mm²) including atypical mitoses and presence of multinucleated cells. SET type HGSCs are characterised by solid sheets of neoplastic cells mimicking endometrioid and/or transitional cell carcinomas, sometimes with bizarre cytologic features. Occasionally a micropapillary pattern can be seen and areas with geographical necrosis can be also found. These tumours are associated with a high number of tumour infiltrating lymphocytes (TILs) [5, 26]. SET pattern is more commonly correlated with germline and/or somatic BRCA1/2 mutations [26] and is more sensitive to chemotherapy and PARP inhibitors.

Based on BRCA mutation status, histomorphological patterns (classic vs. SET), STIC presence and clinical outcome, two categories of HGSC can be identified: (1) BRCA-mutated tumours exhibiting SET morphology, absence of STIC lesions, more common in young patients, more chemosensitive and responsive to PARP inhibitors with favourable prognosis and (2) tumours without BRCA genomic alterations displaying classic morphology, presence of STIC lesions, more common in old aged patients and less responsive to chemotherapy with unfavourable clinical outcome [26, 27].

The WHO 2020 classification introduces specific criteria for site assignment of HGSCs origin (fallopian tube, ovary, tubo-ovarian or peritoneal) Table 1 [5]. According to these criteria, 80% of the cases are of tubal origin, whereas peritoneal origin should be considered only after careful exclusion of STIC and absence of ovarian involvement.

Primary site	Criteria for diagnosis
Fallopian tube	Presence of STIC; or; Presence of invasive fallopian tube HGSC; or; Part or entire fallopian tube length inseparable from tubo-ovarian tumor
Ovary	Both fallopian tubes separable from ovarian tumor; and; No STIC or invasive HGSC in either fallopian tube examined by SEE-FM (sectioning and extensively examining the tubal fimbria)
Tubo-ovarian	Fallopian tubes and ovaries not available for full examination; and; Pathological findings consistent with extrauterine HGSC
Peritoneal	Both tubes and both ovaries fully examined; and; No gross or microscopic evidence of STIC or HGSC in tubes or ovaries

Table 1.

Criteria for assigning primary site in high grade serous carcinomas.

2.2 Immunophenotypic features of ovarian high-grade serous carcinomas

Immunohistochemical analysis should be performed in order to distinguish ovarian high-grade serous carcinoma from mesothelioma or other poorly differentiated carcinomas in cases of peritoneal carcinomatosis. It can also be useful in small biopsies and in post-therapy specimens.

Both classical and SET HGSC histotypes show positive immunoreactivity against CK7, p16, PAX8, WT1, ER (oestrogen receptor) and PgR (progesterone receptor), and this panel of antibodies is enough to establish an ovarian serous carcinoma diagnosis [5]. In addition, 30–50% of HGSCs can exhibit three different aberrant expression patterns for p53, strong, diffuse nuclear overexpression in >80% of cells associated with TP53 missense mutation, no expression implying p53 loss of function and diffuse cytoplasmic expression with weak nuclear intensity similar to wild-type immunoreactivity correlated with loss of function mutations disrupting the nuclear localization signal domain [28, 29]. Strong positive p53 immunoreactivity can also help to identify foci of intraepithelial carcinoma on the ovarian surface or in the fallopian tube epithelium (STIC). HGSCs also exhibit cytoplasmic and/or nuclear p16 expression along with a high Ki-67 (MIB-1) cell proliferation index (>75%) [30].

Ovarian HGSCs can be differentiated from mesotheliomas using a panel of various antibodies such as PAX8, Ber-EP4, MOC-31, ER (positive expression in ovarian serous carcinomas) and Calretinin, CK5/6 (both positive in mesotheliomas) [31].

Diagnostic problem can arise between HGSC of solid morphologic patterns and a poorly differentiated endometrioid carcinoma. In such a case serous carcinoma displays diffuse strong staining for WT1, p53 and p16 [32, 33].

Serous carcinoma of the endometrium shows negative or focally weakly positive WT1 immunoreactivity but p53 positive, therefore, metastatic serous carcinoma with this immunophenotype should be considered endometrial than ovarian origin [33]. WT1 immunopositivity is also observed in serous borderline tumours and in sex cord-stromal tumours [34, 35]. On the other hand, serous borderline and benign tumours show negative immunoreactivity for p53 [29, 33].

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, 48, 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, 55, 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, 58, 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, 61, 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].

3. Low-grade serous ovarian carcinomas (LGSCs)

Low-grade serous carcinomas (LGSCs) are more common in younger women, with a median age of 43 years, have a better clinical course and prognosis than HGSCs and account for approximately 3–5% of epithelial ovarian tumours [67]. LSGCs originate from benign or borderline serous tumours and about 50% are associated with a borderline component. Progression of borderline serous tumour into low-grade serous carcinoma occurs in 6–7% of the cases and evolution to high grade serous carcinoma is rare. A more aggressive clinical course is associated with the presence of a borderline serous tumour component showing micropapillary histological pattern, microinvasions and bilateral ovarian presence [68].

3.1 Pathology of ovarian low-grade serous carcinomas

Macroscopically LGSCs are often bilateral and have a papillary appearance. Foci of calcification may be present. Microscopically, they display papillary, micropapillary, glandular, nested or inverted macropapillary architectural patterns- free-floating within unlined empty spaces, with a variety of invasion patterns. Neoplastic cells demonstrate low to moderate grade nuclear atypia with no pleomorphism (<3× size variation), distinct central nucleoli and relatively low mitotic activity (1–2 mitoses/mm²). Necrosit areas are rare and psammoma bodies are frequent. LGSCs are differentiated from their serous borderline tumour component by the presence of stromal invasive foci measuring >5 mm or 10 mm² in size [5, 69].

3.2 Immunophenotypic features of ovarian low-grade serous carcinomas

LGSCs show positive immunoreactivity against CK7, PAX8, WT1, ER (oestrogen receptor) and PgR (progesterone receptor). Unlike HGSCs, they display patchy or negative expression for p16, low levels of Ki-67 proliferation index (less than 3%) and wild-type immunoreactivity for p53 [70].

3.3 Molecular pathology of ovarian low-grade serous carcinomas

LGSCs are characterised by genomic stability with low mutation rates, are not associated with BRCA germline genomic alterations and display low copy number genetic aberrations, like chromosomal loss of 1p36.33, 9p and homozygous deletions of the CDKN2A/2B locus in approximately 86% of LGSCs [71, 72].

The most common molecular alterations found in LGSCs have activated mutations of upstream regulators of MAPK signal transduction pathway like KRAS (25–54%), BRAF (8–33%), NRAS (8–26%) and ERBB2 (5–6%) [4, 73]. KRAS and BRAF mutations are early events in LGSCs evolution and can be found in 85% of benign cystadenomas and serous borderline tumours [74]. KRAS mutations are associated with aggressive biological behaviour and unfavourable prognosis whereas BRAF mutations are found in early clinical stage [75, 76]. Other driver mutations involved in the pathogenetic mechanisms of LGSCs have been observed in PIK3CA, FFAR1, MACF1 (11%), USP9X (11-27%), ARID1A (9%), NF2 (4%), DOT1L (6%), ASH1L (4%) and EIF1AX (15%) [77]. USPX9 and EIF1AX are downstream effectors of MAPK pathway and linked to the mTOR pathway. Therefore, mTOR inhibitors may be used for targeted therapies in chemoresistant recurrent tumours [78].

4. Ovarian endometrioid carcinomas (OECs)

Endometrioid ovarian carcinomas account for 10–15% of epithelial ovarian tumours are correlated with favourable prognosis and can be found in women ageing 30–80 years [79]. They arise mainly from endometriosis lesions and less frequently from benign or borderline endometrioid ovarian neoplasms, such as adenofibromas or endometrioid borderline tumours [80]. Atypical endometriosis is the precursor lesion for 40% of OECs. Risk factors for OECs are endometriosis lesions, Lynch syndrome, hereditary breast and ovarian cancer syndrome, late menopause and post-menopausal hormone therapy [80, 81].

4.1 Pathology of ovarian endometrioid carcinomas

Endometrioid carcinomas of the ovary present mainly as unilateral mass in the ovary and less frequently as bilateral (20%) [82]. They display a smooth external surface and a solid/cystic cut surface, sometimes with a residual endometriotic cyst at the periphery of the tumour [80]. Microscopically, these tumours show a variety of morphologic patterns such as glandular, cribriform, villoglandular or solid with characteristic back-to-back glands, areas of squamous differentiation and expansile rather than an infiltrative pattern of invasion [5]. Sometimes a destructive invasion pattern can be seen characterised by neoplastic cells infiltrating the stroma accompanied by a desmoplastic reaction. The neoplastic cells are tall columnar and focally mucinous with a mitotic activity of 5–10 mitoses per high power field [5, 82]. Histologic characteristics confirmatory of OECs are metaplastic features such as squamous, morular, hobnail or mucinous metaplasia, presence of endometriosis lesions, ovarian endometrioid adenofibroma or endometrioid borderline tumour, and presence of a synchronous uterine endometrioid carcinoma found in 15–20% of the cases [5]. Ovarian endometrioid carcinomas are divided according to the presence of solid growth pattern in grade 1 (less than 5% solid growth), grade 2 (5–50% solid growth) and grade 3 (more than 50% solid growth), excluding areas of squamous differentiation [5].

4.2 Immunophenotypic features of ovarian endometrioid carcinomas

OECs show diffuse immunopositivity for CK7, PAX8, ER and PgR [83]. Approximately 33% of OECs display membranous and/or cytoplasmic diffuse expression for vimentin [84]. Squamous morules of endometrioid tumours demonstrate strong CD10 cytoplasmic expression [85] and infrequently CDX2 immunopositive reaction [86]. Endometrioid ovarian carcinomas also exhibit β-catenin membranous and/or cytoplasmic expression. Nuclear β-catenin expression is correlated with CTNNB1 genomic alterations and favourable prognosis [77]. Differential diagnosis between high-grade endometrioid carcinomas with solid architectural patterns and high-grade serous carcinomas can be made based on the intense diffuse immunopositivity of WT1, p53 and p16 in HGSCs, whereas ECs display patchy expression for p16, wild-type or mutation-type expression for p53 and loss of WT1 immunoreactivity [87].

4.3 Molecular pathology of ovarian endometrioid carcinomas

OECs are characterised mainly by mutations in PIK3CA (15–40%), ARID1A (30–35%), a component of the SW1/SNF chromatin remodelling complex, KRAS (10–30%) and CTNNB1 (25–60%) involved in the WNT/β-catenin signal transduction pathway [88, 89, 90]. Borderline endometrioid ovarian tumours have CTNNB1 mutations in 90% of the cases [77, 90]. Other less common genomic alterations are mutations in PTEN (20–30%) with frequent loss of heterozygosity (45–75%), TP53 (10–25%) and POLE (3–10%) [91, 92]. Somatic or germline predisposition mutations in MMR genes (MLH1, MSH2, MSH6, PMS2) can be found in 10–20% of the cases, some of them associated with Lynch syndrome [93, 94]. POLE-mutated EOCs and MMR-deficient ovarian endometrioid tumours have favourable clinical outcomes [95, 96]. CTNNB1-mutated tumours show low genomic complexity and are correlated with low-grade tumours and good prognosis, unlike their uterine endometrioid counterparts, which demonstrate worse clinical outcomes [77, 97]. TP53 mutated EOCs have high genomic complexity with poor prognosis [98]. Synchronous presence of endometrial and ovarian endometrioid carcinomas can be encountered in 25% of the cases demonstrating a putative clonal relationship with favourable prognosis [99, 100]. Ovarian seromucinous carcinomas (mixed serous and endocervical-type mucinous carcinomas) are considered by the current WHO classification a subtype of endometrioid ovarian carcinomas based on their morphological and molecular overlapping features [5, 101, 102]. Based on their molecular features, EOCs are divided into four molecular subcategories: 1. hypermutated with microsatellite instability due to MMR deficiency (10–20%), 2. ultramutated due to POLE exonuclease domain mutations (3–10%), 3. TP53 mutated (10–25%) and 4. with no specific molecular signatures (60–70%) [95, 103]. Hypermutated and ultramutated EOCs display high mutation burden, a molecular finding that might be exploited for immunotherapeutic interventions. The genomic aberrations identified in EOCs might be used as targets for therapeutic interventions, such as targeting mutated ARID1A with HDAC inhibitors or targeting dysregulated MAPK or PI3K pathways using MEK or PIK3AC inhibitors, respectively [104, 105].

5. Ovarian mucinous carcinomas (OMCs)

Primary ovarian mucinous tumours are relatively rare, approximately 10–15% of all ovarian tumours and most of them (80%) are benign or borderline mucinous tumours. Mucinous carcinomas in the ovary represent 3% of ovarian carcinomas with relatively high prevalence in women below 40 years of age [106] and most of them are of metastatic origin, particularly from the gastrointestinal or pancreatobiliary tract associated with pseudomyxoma peritonei [5, 107]. Primary OMCs originate mainly from mucinous benign or borderline tumours, with a small percentage originating from mature cystic teratomas or Brenner tumours with gastrointestinal pattern component [106, 107]. They are more commonly unilateral of large size (>13 cm) with no ovarian surface involvement. On the other hand, metastatic mucinous carcinomas are bilateral, smaller in size and associated with pseudomyxoma peritonei and imaging findings from other organs, mainly gastrointestinal or pancreatobiliary tract. They have a favourable prognosis in early clinical stages, albeit in advanced stages they show chemoresistance with unfavourable clinical outcomes [108, 109].

5.1 Pathology of ovarian mucinous carcinomas

Ovarian mucinous carcinomas are usually unilateral masses, large in size (8–40 cm), with the presence of unilocular or multilocular cysts filled with mucinous content. These tumours may display a variety of lesions from cystadenoma areas to borderline mucinous tumour regions and carcinomatous components. Microscopically there are two major histotypes, the intestinal and the endocervical. The intestinal type is more common than the endocervical. They exhibit glandular, cribriform, papillary and solid patterns of growth with two different patterns of invasion (i) expansile/confluent (more common) with a back-to-back glands labyrinthine complex appearance and minimal stroma and (ii) infiltrative/destructive characterised by irregular malignant glands infiltrating desmoplastic stroma. Each pattern of invasion measures 5 mm or more in linear size and they may coexist [5, 108]. Rarely, mural nodules of anaplastic carcinoma or high-grade sarcomatous-like component may be seen in ovarian mucinous carcinomas [5, 110]. There is no standardised grading system for OMCs till now.

5.2 Immunophenotypic features of ovarian mucinous carcinomas

Immunohistochemistry of OMCs is characterised by diffuse intense positivity for CK7 and variable positivity for CA19-9, CEA, CK20 and CDX2 focal weak positivity for PAX8 can be seen in a subset of tumours [111, 112], whereas WT1, Napsin A, Vimentin, CA125, ER and PgR are mostly negative. SATB2 is rarely expressed in ovarian mucinous tumours (5–7%) and its expression is associated with the presence of mature teratoma [113] p53 may demonstrate wild-type or mutation-type immunoreactivity and p16 is usually negative or focally positive [114, 115].

5.3 Molecular pathology of ovarian mucinous carcinomas

OMCs are characterised by KRAS and TP53 mutations (64–66%), CDKN2A inactivation (76%) and HER2/neu gene amplification (20–26%) [107, 116]. HER2 gene amplification is almost mutually exclusive to KRAS mutations and is found in most of the cases with mutated TP53 [64%) [116]. OMCs can be developed from benign mucinous tumours through a progression tumour evolution model starting with KRAS or CDKN2A genomic alterations. Both KRAS and CDKN2A mutations along with extra genomic copy number aberrations have been found in mucinous borderline tumours and, therefore, are regarded as early molecular events [117, 118]. Chromosomal locus 9p13.3 amplification and TP53 mutations are identified at the final evolutionary steps of OMCs’ progressive carcinogenesis [118]. Other less frequently mutated genes in MOCs are PIK3CA, PTEN, BRAF, CTNNB1/APC (regulators of the β-catenin/Wnt signal transduction pathway), RNF43 and ARID1A (8–12%) [107]. About 34% of OMCs have neither KRAS nor HER2 gene alterations and are considered to be neoplasms arising from mature cystic teratomas and correlated with an increased risk of recurrence and poor clinical outcome [119]. OMCs with high number of genomic aberrations and mutational burden are associated with high grade and unfavourable prognosis [107]. Targeted therapeutic approaches against HER2 amplification and/or MAPK pathway mutations might be applied along with other inhibitors, such as HDAC inhibitor for ARID1A, for more effective tailored treatment of OMCs [120].

6. Ovarian clear cell carcinomas (OCCCs)

Ovarian clear cell carcinomas comprise 10–12% of ovarian carcinomas with relatively high prevalence in young women of East Asian origin [121]. They are frequently associated with endometriosis lesions (50–74% of the cases) and/or clear cell benign (adenofibroma) or borderline tumours, pathologic entities with PIK3CA and ARID1A precursor genetic alterations [122]. They present mostly as unilateral mass in clinical stage I or II [5] and are considered a high-grade malignancy, although stage I patients have a favourable prognosis. Advanced-stage patients are related to poor clinical outcomes due to chemoresistance. Predisposition risk factors are late menopause, Lynch syndrome and expression of the genetic locus HNF1B through epigenetic mechanisms [122]. Lynch syndrome-associated OCCCs or tumours with MMR deficiency are correlated with long survival due to putative tumour immunogenicity [123].

6.1 Pathology of ovarian clear cell carcinomas

Grossly, OCCCs are large (mean size 13 cm) unilateral masses, solid and cystic in appearance, frequently containing endometriosis lesions. Microscopically, they have solid, tubulocystic and papillary architectural patterns and are composed of neoplastic cells characterised as polygonal, cuboidal or hobnail-like cells with clear cytoplasm, atypical nuclei and distinct nucleoli, without prominent pleomorhism [5, 122]. Mitotic activity is less than 5 mitoses per 10 high power fields. The clear cytoplasm is glycogen rich, PAS positive and diastase sensitive. Stromal hyalinization and myxoid appearance can be seen frequently [5].

6.2 Immunophenotypic features of ovarian clear cell carcinomas

OCCCs are strongly immunopositive for Napsin A (80–85%) and HNF1β (80–92%) [124, 125] and negative for WT1, ER and PgR. In addition, OCCCs show a wild-type pattern of p53 expression [126]. Differential diagnosis of OCCCs should be made among ECs and HGSCs because in bothtumours clear cell change can be observed, HGSCs demonstrate positive immunoreaction for WT1 and ER while ECs show positivity for ER [127]. Additionally, both tumours (HGSCs and ECs) are negative for napsin and HNF1β [127].

6.3 Molecular pathology ovarian clear cell carcinomas

The genomic aberrations found in OCCCs involve PIK3CA activating mutations (40–50%), ARID1A loss of function mutations (50–75%), MET gene amplifications, mutations in ARID1B (10%), KRAS (15%), PPP2R1A (15%), TERT promoter (15%), SMARCA4, PTEN (1–5%), PIK3CA, PIK3R1, AKT2, TP53 (5–20%) and ZNF217transcription factor overexpression, which is associated with poor outcome [128, 129]. PIK3CA mutations commonly coexist with ARID1A genomic alterations and are more frequent in endometriosis-associated OCCCs [128]. Studies on genes involved in antioxidant cell machineries such as GPX3 (glutathione peroxidase 3), GLRX3 (glutaredoxin) and SOD3 (superoxide dismutase) have shown that these genes are highly expressed in CCOCs resulting in tumour chemotherapy resistance [129, 130]. HER2 gene amplification (15%) and Mismatch Repair (MMR) gene deficiency (2–3%) have also been identified [131]. MMR germline mutations can be found in 10% of OCCCs and may predispose them in developing OCCCs [131]. Therefore, MMR gene expression should be tested by immunohistochemical methods or by MSI testing in order to identify OCCCs associated with MMR deficiency and/or Lynch syndrome. This is important, taking into account that MMR deficient OCCCs are correlated with favourable clinical outcome even in advanced stages [132]. Mutations of the TP53 gene are usually rare, albeit abnormal p53 expression (7%) has been reported and is associated with adverse prognosis [132]. Molecules involved in the PIK3/AKT/mTOR pathway and loss of function of ARID1A gene might be targeted therapeutically by using mTOR inhibitors and through inhibition of EZH2 transcription factor, respectively [133].