Characterization of ovarian clear cell carcinoma using target drug-based molecular biomarkers: implications for personalized cancer therapy

The histological types of epithelial ovarian carcinoma (EOC) have distinct clinical and molecular features with each other [1, 2]. To date, EOC patients are mostly provided with the same therapeutic regimens regardless of the distinct histological characteristics of the primary lesion [3]. However, ovarian clear cell carcinoma (CCC), contributing for 4.8–25% of all ovarian carcinomas [4], is known to be less sensitive to platinum-based front-line chemotherapy and to be associated with a poorer prognosis than the more common serous subtype. Moreover, previous studies have shown that the response rate of various second-line chemotherapeutic regimens for recurrent platinum-resistant ovarian CCC was only 1% [5]. Therefore, a further investigation of the mechanism of the chemoresistance and developing new therapeutic targets are needed for effective clinical management of ovarian CCC.

Although the mechanism of carcinogenesis and chemoresistance of ovarian CCC is still unclear, several genetic changes have been widely investigated. Compared with ovarian high-grade serous carcinomas (HGSC), ovarian CCC are generally negative for p53 mutation and have a lower frequency (6.3%) of breast cancer 1 or 2 (BRCA1/2) mutations [6, 7]. A higher frequency of AT rich interactive domain 1A (ARID1A) and phosphatidylinositol-4, 5-bisphosphate 3-kinase catalytic subunit α (PIK3CA) mutations (36%) were also observed in CCC patients [8, 9].

In addition, protein expression studies of ovarian CCC observed five functional protein activating pathways [10], some of which could be potential therapeutic targets: (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway, hypoxia-inducible factor 1α (HIF-1α)/vascular endothelial growth factor (VEGF) pathway, hepatocyte nuclear factor 1β (HNF-1β) pathway, interleukin 6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) pathway and MET pathway [11‐15]. Recently, Sereni et al. detected 117 drug-based protein expression levels of 72 EOC patients by using reverse-phase protein microarray (RPPA) analysis. They found that 11 out of 117 proteins of ovarian CCC (n = 7) showed a distinct signaling network from other subtypes of EOC, such as enhanced activation of phosphorylated epidermal growth factor receptor (EGFR) and phosphorylated human epidermal growth factor receptor-2 (HER2) [16]. However, the sample size of ovarian CCC in the study of Sereni et al. was not large enough due to the rarity of this subtype. In the current study, we simultaneously compared seven drug-based protein expression levels (either in an existing FDA-approved therapy or in an experimental therapeutic in clinical trial) both in ovarian CCC and HGSC on a larger scale and investigated their associations with clinical characteristics and survival outcomes.

Deciphering ovarian CCC molecular expression patterns is important not only for understanding the pathogenesis of this rare disease but also for exploring therapeutic options for patients who are chemotherapeutic resistant. In this study, we investigated the molecular characterization of CCC patients compared with HGSC patients on several actionable drug-based molecular biomarkers. These molecular biomarkers might be valuable for predicting the outcome and the response to chemotherapeutic regimens for CCC, compared with the classical clinicopathological prognostic factors [17].

EGFR and HER2 are the members of the HER family, whose EGF signaling pathway is known to play a crucial role in tumor initiation, progression and metastasis. In this study, EGFR and HER2 protein were more frequently expressed in CCC patients than in HGSC patients (35.9% vs 27.4%, 12.6% vs 2.7%, respectively). Besides, Aikou Okamoto et al. observed a gene amplification of EGFR in CCCs and it was related with endometriosis [18]. In line with this, our finding showed that there was 44.4% CCC patients with endometriosis expressed high level of EGFR, while only 8.3% patients with endometriosis in HGSC have high expression of EGFR (Fig. 4, P = 0.049). Tan et al. also reported that HER2 gene amplification and protein overexpression were observed in 14% of CCCs through hierarchical cluster analysis [19]. However, Friedlander ML et al. demonstrated that only 2.6% of pure CCCs expressed high level of HER2 protein by IHC tests, but 9.4% of CCCs showed HER2 gene amplification by fluorescent in situ hybridization (FISH) tests [20]. These inconsistent results might be caused by differences in the scale of cohorts and criteria of the cut-off value for positive cases. Moreover, evidence on the prognostic value of EGFR and HER2 with respect to survival is still inconclusive in ovarian cancer [21]. We observed a favorable prognostic value of EGFR in CCCs and a reverse prognostic value in HGSCs. This contrasting result could be explained by the phenomenon that the positive rate of EGFR of CCC patients in early stage (I + II) was much higher than of HGSC patients in early stage (39.7% vs 17.7%, Additional file 1: Table S2A and B), which implied that EGFR might play a favorable part in the earlier progression of CCC. And previous studies either focused on stage III/IV ovarian patients, or performed IHC assay in a smaller sample size of CCC because of the rarity of this subtype, which might cause bias. In addition, Wang et al. showed that high expression of EGFR in tumor stroma, rather than in tumor cells, correlates with aggressive clinical features in epithelial ovarian cancer, and is an independent prognostic factor [22]. This complexity of the role of EGF signaling pathway in ovarian cancer explained, at least in part, why several clinical trials performed on patients with positive EGFR or HER2 immunostaining or genetic changes did not show satisfying response to HER-targeted therapy [23‐25].

TCGA (The Cancer Genome Atlas) analyses showed that the BRCA1 and BRCA2 mutations in 22% of the high-grade serous ovarian cancer samples triggered a wide range of aberrations in DNA damage repair pathways, such as homologous repair pathway [26]. Patients with germline BRCA mutation gained great clinical benefit from using PARP (Poly ADP-ribose polymerase) inhibitors, olaparib for instance, in clinical trials [27]. Beyond this, Konstantinopoulos et al. found that BRCAness profile (patients with sporadic cancers that have a BRCAness phenotype, characterized by defective homologous repair) correlates with responsiveness to platinum and PARP inhibitors and identifies a subset of patients with improved outcome [28‐30]. Our analysis revealed that loss of BRCA1 expression was more frequently occurred in HGSC patients than in CCC patients, predicting a shorter overall survival time (P = 0.017), which implied potential benefits of PARP inhibitors. However, it is suggested a deeper exploration of the relationship between genetic changes and protein expression of BRCA1/2 in sporadic ovarian cancer. In addition to somatic BRCA1/2 mutation, PTEN loss may be a common contributing event causing homologous repair dysfunction. Previous studies have shown that ovarian CCC often exhibit genetic alterations of PTEN in 5% CCC patients [31], and preclinical researched showed that ovarian cell lines with PTEN mutation were more sensitive to PARP inhibitors [32]. In the present study, we observed a much higher level of PTEN loss in both of HGSCs (87.6%) and CCCs (89.2%), compared with previous studies [17, 20]. This might be due to the different efficacy of the antibodies used in IHC test. According to our analyses (Additional file 1: Table S2A and B), CCCs have a higher rate of PTEN silence in early stage than HGSCs (92.0% vs 70.5%, respectively). However, targeting PTEN only is not enough due to tumors with ARID1A mutation also frequently harbor PTEN or PIK3CA mutation in CCC [33, 34]. Therefore, it is worth trying to use PARP inhibitors for patients with sporadic cancers who lose BRCA or PTEN expression, as well as in combination with other target drugs if necessary.

Aurora kinase A (AURKA) is involved with centrosome function, mitotic entry and spindle assembly in cells [35]. Recently AURKA has been extensively investigated in different neoplasms, including breast, ovarian, colon, liver, pancreas, bladder, and gastric carcinomas [36]. Even if the molecular mechanism of AURKA is complex [37, 38], several researches implied that AURKA protein expression is strongly linked with poor patient outcome and aggressive disease characteristics of ovarian cancer [39, 40]. Consistently, we found the same effects of AURKA in CCC patients that both of the overall survival time and progression-free time were shortened in CCCs with high expression level of AURKA (P < 0.05, Table 3). In addition, overexpression of AURKA also had a relationship with platinum-based chemotherapeutic resistance in CCC patients, suggesting that targeted at AURKA might provide a promising therapeutic option. Most recently, ENMD-2076, acting by blocking AURKA and tyrosine kinase enzymes from working and stopping new blood vessels from growing, is in phase II clinical trial designed specifically for CCC patients (ClinicalTrials.gov number, NCT01914510). The result will be posted in 2018.

Surgery and chemotherapy is the mainstay of ovarian cancer treatment. However, with the development of immunology, immunotherapy represents a rational and alternative approach for ovarian patients, especially for those who have recurrent and/or chemotherapeutic resistant diseases [41‐43]. Programmed death 1 (PD-1) and its ligand PD-L1 play a crucial role in the inhibition of T cell-mediated immune response. Binding of PD-L1 to PD-1 causes the exhaustion of effector T cells and immune escape of tumor cells, leading to poor prognostic outcome. Previous studies have shown that overexpression of PD-L1 in cancers such as gastric cancer, hepatocellular carcinoma, renal cell carcinoma and esophageal cancer, pancreatic cancer and bladder cancer is related to poor prognosis. However, PD-L1 expression correlates with better clinical outcomes in breast cancer and Merkel cell carcinoma [44]. The prognostic value of PD-L1 in ovarian cancer is controversial [45, 46]. A recent study of 490 EOC cases revealed that PD-L1 had a positive association with survival in high-grade serous subtype rather than clear cell subtype [45]. Accordingly, our results also showed that PD-L1 expression was correlated with a favorable prognosis of HGSC patients (Fig. 2, P = 0.037), which might be caused by an immunological stalemate that activation of T cells triggered a negative feedback in the microenvironment around tumor cells. Another study reported by Friedlander ML et al. showed that only 7.4% of CCC cases have aberrant PD-L1 overexpression through IHC method. However, the sample size in Friedlander’s study is too small (n = 27) and no detailed clinical information to further analyze [20]. By performing analysis on a larger scale (n = 96), we found that there was no significant correlation between PD-L1 expression and prognosis in CCC patients, but higher expression of PD-L1 was observed in this subtype than in HGSC (21.1% vs 11.6%, respectively). And by combining with the analysis of chemotherapeutic response, it is revealed that CCC patients with high expression level of PD-L1 tend to be more resistant to platinum-based chemotherapy (P = 0.028), which suggested that PD-L1 might play a part in the chemoresistance of CCC patients.

It is true that different studies may probably draw different conclusions from TMA immunohistochemistry (IHC) because of various reasons, such as the efficacy of antibodies, intra-tumor heterogeneity, criteria of the cut-off value for positive cores and sufficient follow-up time of patients. Although we have tried our best to balance these factors, there are still some limitations in our study. Firstly, since there being no uniform interpretations for IHC scoring and analyses, we used the same cut-off value for all antibodies, which might not be optimal for each marker. Secondly, we did not perform correction for multiple comparisons in some of our analyses. While IHC assay could only detect protein expression changes, DNA sequencing is still needed to further investigate genes status for these molecular biomarkers. Indeed, our work is exploratory and descriptive, we would try to solve these problems and perform larger cohort studies combined with genomic changes in the next future.

Ovarian clear cell carcinoma showed a markedly different biomarker protein expression map from high-grade serous carcinoma. Expression level of HER2 and PD-L1 were higher in CCC patients, compared with HGSC. Besides, overexpression of AURKA and PD-L1 significantly associated with chemoresistance in CCC. All these differences suggested newly designed clinical trials of candidate target drugs for this rare subtype, and deserved further investigation of potential molecular mechanisms.