Epithelial ovarian cancer is classified into four major subgroups: serous, clear cell, endometrioid and mucinous (1). Ovarian clear cell carcinoma (OCCC) has phenotypes distinct from those of the other subgroups, such as resistance to chemotherapy, poor prognosis, an association with endometriosis, a higher incidence of thrombosis as a complication and a higher incidence among Japanese individuals (2–4). OCCC contains mutations in various tumor suppressors and oncogenes, including AT-rich interactive domain-containing protein 1A (ARID1A), phosphatidylinositol-3-kinase catalytic subunit (PIK3CA), KRAS and TP53 (5). Furthermore, a gain in DNA copy number is frequently found in chromosome 20, which contains the potential oncogene zinc finger protein 217 (ZNF217), while losses in DNA copy number are observed at the CDKN2A/2B and LZTS1 loci (6,7).

We recently analyzed somatic copy number alterations in OCCC using comparative genomic hybridization (CGH) (8). In agreement with previous reports (7), we found recurrent amplification of the 20q13 locus containing ZNF217 and the 17q23.2 locus harboring PPM1D. Furthermore, we found recurrent amplification of the entire chromosome 8q, the EGFR and HER2/ERBB2 gene loci, and losses of chromosomes 9q, 13q and 17q. In the present study, we reanalyzed this CGH data and further identified amplification in the chromosomal regions 8p11.21–22 and 12p13.31 as well as 20q13.2, and losses of small segments in the chromosomal regions 3q26.1, 4q13.2 and 22q11.23 in OCCC. Moreover, we showed that these DNA copy number alterations are associated with poor patient prognosis.

The characteristics of the 110 patients (95 Japanese and 15 Korean patients) studied are summarized in Table I. With a median follow-up of 17.8 months (range, 10–36 months), 33 (30%) patients relapsed and 21 (19%) patients died. The mean age was 53 years (range, 30–86 years). FIGO stages were: stage I, 61 patients; stage II, 14 patients; stage III, 32 patients; stage IV, 3 patients. Endometriosis was diagnosed in 54 (49%) patients. Thrombosis was observed in 12 (11%) patients. The response to chemotherapy was: platinum sensitive, 60 (55%) patients; platinum resistant, 26 (24%) patients.

In our previous CGH analysis of somatic copy number alterations in OCCC (8), we selected regions with a minimum average log₂ ratio ±0.25 (8). In the present study, we reanalyzed the CGH data from 110 OCCC patients: regions with an average log₂ ratio ≥0.9 or ≤-1.5 were defined as those with a copy number gain or loss, respectively (Figs. 1 and 2). We identified increases in DNA copy number of the chromosomal region 8p11.22, which harbors ADAM5p and ADAM3A, in tumor tissues from ~25% of patients; 8p11.21, harboring a number of genes, including ANK1, MYST3/KAT6A, IKBKB, POLB and DKK4, in ~14–16% of patients; 12p13.31, harboring the long non-coding RNAs (lncRNAs) ENST00000518709 and ENST00000545710, in ~13% of patients; and 20q13.2, harboring the candidate oncogene ZNF217, in 15% of patients (Fig. 1 and Table III). In addition, we identified losses of small segments (regions with an average signal ratio ≤-0.5) in the chromosomal regions 8p11.22 and 12p13.31 in normal samples from 5 and 17% of OCCC patients, respectively.

We also identified losses of small segments in the chromosomal region 3q26.1, which harbors the lncRNA BC073807; 4q13.2, harboring UDP-glucuronosyltransferase 2B17 (UGT2B17); and 22q11.23, harboring glutathione S-transferase θ 1 (GSTT1) and D-dopachrome tautomerase-like (LOC391322), in tumor tissues from 33, 19 and 4% patients, respectively (Fig. 1 and Table III). In addition, we identified losses of small segments in the chromosomal regions 3q26.1, 4q13.2 and 22q11.23 in normal samples from 45, 36 and 24% patients, respectively.

To validate these results, we performed ddPCR analysis of copy number for ANK1, MYST3, IKBKB and POLB in four OCCC patients (Fig. 4, left panels). Consistent with the results of the CGH analysis, these genes were amplified in three OCCC patients with 8p11.21 amplification, OC19, OC69 and OC96, but not in a patient without 8p11.21 amplification, OC20 (Fig. 3). We also confirmed that ZNF217 was amplified in a patient with 20q13.2 amplification, OC20. Analysis of 9 patients confirmed the amplification of these genes (Fig. 4; 4 patients in the left panels and 5 patients in the right panels). ADAM5p and the lncRNA ENST00000518709 appeared to be amplified, but no statistical significance was found (Fig. 4). In addition, consistent with the results of CGH analysis, we observed losses of ADAM5p and the lncRNA ENST00000518709 in normal samples in 3 out of 10 and 7 out of 9 OCCC patients, respectively. Furthermore, we performed exome sequence analysis of 3 patients and confirmed that 8p11.21, but not 20q13.2, was amplified in OC96 (Fig. 5 and Table IV).

We finally investigated the relationship between the amplification or loss of these genes and survival, sensitivity to chemotherapy, thrombosis and endometriosis. The Kaplan-Meier survival analysis showed that patients with amplification of chromosome 8p11.21 had shorter PFS than those without such amplification (P=0.057, 95% CI: 0.961–5.154) (Fig. 6A). Patients with amplification in three of the four chromosomal regions 8p11.21, 8p11.22, 12p13.31 and 20q13.2 had shorter OS (P=0.068, 95% CI: 0.833–16.393) as well as shorter PFS (P=0.001, 95% CI: 1.615–13.698) (Fig. 6B and C, Table V). Patients with a deletion in the chromosomal region 3q26.1 had shorter PFS (P=0.046, 95% CI: 0.998–4.000) (Fig. 6D). Analysis of the correlation between copy number alterations and sensitivity to chemotherapy revealed that amplification of 12p13.3 or three of the four chromosomal regions 8p11.21, 8p11.22, 12p13.31 and 20q13.2 was associated with resistance to platinum-based chemotherapy (P=0.011 or 0.012, respectively) (Table VI). We also found that a deletion in the chromosomal region 3q26.1 was associated with chemotherapy resistance (P=0.008) (Table VI). We did not observe any association between copy number alterations and sensitivity to chemotherapy, FIGO stage, thrombosis or endometriosis.

In the present study, we showed that the chromosomal regions 8p11.21, 8p11.22 and 12p13.31 are amplified and the chromosomal regions 3q26.1, 4q13.2 and 22q11.23 are deleted in OCCC. Kaplan-Meier survival analyses revealed that patients with amplification within 8p11.21 or a deletion within 3q26.1 had a shorter PFS than those without such copy number alterations. Furthermore, patients with amplification in three of the four chromosomal regions 8p11.21, 8p11.22, 12p13.31 and 20q13.2 had shorter OS. Consistent with these results, we found that amplification of 12p13.3 or three of the four chromosomal regions 8p11.21, 8p11.22, 12p13.31 and 20q13.2, or a deletion in the chromosomal region 3q26.1 is associated with chemotherapy resistance (Table VI). Thus, amplification in 8p11.21, 8p11.22, 12p13.31 and 20q13.2 and a deletion in the chromosomal region 3q26.1 may be critical for the survival of OCCC patients.

The chromosomal region 8p11.21 encodes a number of genes, including ANK1, MYST3, IKBKB and POLB, which have been reported to play important roles in the development of a number of cancers (13,14). It is therefore possible that amplification of the genes encoded in this region contribute to the development of OCCC.

ADAM5p and ADAM3A encoded in the chromosomal region 8p11.2 are pseudogenes of the ADAM metallopeptidases (15). It has recently been reported that pseudogenes can function as lncRNAs, which can play important roles in tumorigenesis. For example, PTENP1 functions as a competing endogenous RNA to suppress tumor progression (16). UPAT, which is encoded by the pseudogene of the amine oxidase copper containing-3 (AOC3) gene, interacts with and stabilizes the epigenetic factor UHRF1 by interfering with its β-TrCP-mediated ubiquitination, thereby promoting the tumorigenicity of colorectal cancer cells (17). In addition, it has been reported that the chromosomal region 8p11.22 is amplified and ADAM5p and ADAM3A are overexpressed in conjunctival squamous cell carcinoma and glioma (18,19). We also found that the lncRNA ENST00000518709 is amplified in OCCC patients. Thus, it is possible that amplification of these lncRNAs is important for the development and progression of OCCC. In addition, we observed losses of 8p11.22 and/or 12p13.31 in normal samples from 5 and 17% of OCCC patients, respectively. The significance of these losses in normal tissues remains to be investigated.

While we also found that the chromosomal regions 3q26.1, 4q13.2 and 22q11.23 were deleted in 30, 19 and 4% of OCCC, respectively, we also identified losses of small segments in these chromosomal regions in normal samples from 45, 36 and 24% of patients, respectively. CGH analysis did not discriminate between the segments deleted in normal samples and those deleted only in tumor tissues. These loci encode the lncRNA BC073807, UGT2B17 (a member of the uridine diphosphoglucuronosyltransferase protein family), and GSTT1 and LOC391322, respectively. It remains to be investigated whether deletion of these genes contributes to the development of OCCC. It is also possible that deletion of these regions may disrupt the three-dimensional networks of chromosomal interactions and thereby lead to altered gene expression (20–22). Elucidation of the functional significance of these deletions in the development of OCCC is underway in our laboratories.

In conclusion, we identified somatic copy number alterations that have prognostic value in patients with OCCC. We also identified copy number alterations associated with chemotherapy resistance. However, it remains to be confirmed whether amplification of these genes indeed results in changes in the expression of encoded proteins. Furthermore, it remains to be seen whether amplification or deletion of these genes contributes to the development, progression and/or chemosensitivity of OCCC. The identification and characterization of these genes could provide novel insights into the mechanisms of OCCC development and chemosensitivity. We speculate that these genes could be promising molecular targets and/or markers for OCCC therapy.