Introduction
Among the different gynecologic cancers, ovarian cancer (OC) is the leading cause of death (1). In comparison to the breast cancer, OC mortality rate is approximately three times higher despite its lower incidence rate (2). This is attributed to its asymptomatic initial stages and late diagnosis at advanced stages (3,4). The incidence of OC in Saudi Arabia has increased 4-fold between 1990 and 2016(5). It affects more than 3% of Saudi women in their lifetime (6,7). There are different types of ovarian malignancy; however, the epithelial ovarian cancer (EOC) is the most common malignancy (90%) (4,8-10). This EOC is a heterogeneous disease with several histologic subtypes that exhibited distinct cytogenetic features, molecular signatures and onco-pathological signaling pathways. It is a highly invasive with five distinct histologic subtypes: High grade serous which accounts for 70% of EOC, endometrioid constitutes 10%, clear cell (10%), low grade serous 5% and mucinous 3% of EOC (5).
With huge milestones being achieved in the genomic analysis, some inherited mutations of specific genes, such as BRCA1 and BRCA2, have been shown to increase OC risk. In fact, genomic events such as amplification (HER2/KRAS) and mutations (KRAS, PIK, RAD, BRAD) have been reported in OC (11-13). In contrast, oral contraceptive pills, plurality, hysterectomy, and breastfeeding have lower OC incidence (14,15).
Studies have shown that the 5-year survival rate of patients with OC depends mainly on the disease stage at diagnosis. In general, patients diagnosed at an early stage (I/II) have higher 5 year survivals than those at late stage (III/IV). Furthermore, a noticeable increase in OC post-relapse survival was reported over the years during the last decades (16). With the advent of high-throughput sequencing technologies, the overall aim of precision medicine for patients with OC is to predict, prevent, and treat the disease while improving the clinical outcomes and reducing the adverse effects associated with invasive therapeutic approaches (17). Therefore, there is a need for devising new effective strategies based on genomic and epigenomic signatures to identify relevant molecular biomarkers and establish personalized therapeutics (18,19).
Genomic instability is one of the main causes of cancer onset attributed to the interactions among a patient's susceptible genome, environment, and lifestyle; hence, some DNA alterations occur at the epigenomic level. Epigenetic alterations encompass histone acetylation, noncoding RNAs, and CpG island methylation, are among the most frequent epigenetic alterations observed in OC (20-22). Currently, there is an increasing interest in examining the specific patterns of hypermethylation of CpG islands in OC. Both targeted and whole-genome analyses for DNA methylation patterns have shown potential in identifying methylation biomarkers and their prognostic value in OC (23-26). Moreover, epigenetic silencing of some genes, including hMLH1, RASSF1A, and FANCF due to methylation is associated with ovarian tumor drug resistance (27). Consequently, some key epigenetic events are likely to provide cancer cells an advantage to proliferate and invade, thereby promoting metastasis (28,29). Thus, further studies are required to analyze the methylation profiles of several genes to gain deeper insights into the epigenetic alterations in OC, especially in the Arabian Peninsula, where only a few studies have been reported (25,26,30-32). In this context, klotho (KL) gene is an interesting candidate aging marker gene that could be associated with aging-related diseases e.g. cancer. In fact, KL gene was reported with an aberrantly methylated promoter reported in several malignancies (33). Initially discovered as an anti-aging factor (34), KL is located in chromosome 13q12. It is 50 kb in length and is composed of five exons and four introns (35). It is highly expressed in the kidneys and brain but can also be expressed in the placenta, breast, ovaries, uterus, and fallopian tube tissues (36). In the female reproductive system, particularly in the ovary, the level of abnormal expression of KL in granulosa cells is tightly linked to the severity of ovary-related diseases (37). KL expression levels decrease in the brain with advancing age in mammals. This decrease could be due to hypermethylation of the promoter region of KL (38,39). Furthermore, aberrant methylation in the promoter region of KL decreases gene transcription (40). Recently, KL has been reported as a potential tumor suppressor gene that inhibits the IGF-1 pathway and is a novel target gene for epigenetic silencing in cancers of breast (41), cervix (34), stomach (42), bladder, and ovaries (43). However, low expression levels of KL are associated with weakness in skeletal muscle, difficulty in executing daily activities, and increased mortality in the elderly. Deficiency in IGF-1 expression level is a reported feature of aging, and is likely affected by methylated KL (40). The 1439-bp long promoter region of KL lacks a TATA box and instead, contains four potential Sp1 binding sites (35,44). Moreover, KL expression can be restored using demethylation reagents (35). So far, only few studies have assessed the methylation profiles of KL in OC and showed potential prognosis value. In that, higher KL promoter methylation profiles were associated with cancer progression (45,46). Therefore, additional studies are required to further understand the prognosis value of KL methylation status in OC, particularly, in the Arabian Peninsula population. In this study, we analyzed KL gene promoter methylation profiles in OC and assessed its prognostic value.
Discussion
OC is one of the deadliest gynecological cancers mainly because of delayed diagnosis and asymptomatic early-stage progression. Moreover, the deleterious effects of OC can be attributed to the anatomical position of the ovaries within the deep pelvic cavity which may hide the disease expansion and delay the diagnosis (49-51). Scientists are seeking to improve the detection, prognosis, management, and treatment approaches toward precision oncology to alleviate the burden of this disease on patients, their families, and the healthcare system (52). With the development of high-throughput sequencing technologies, it is possible to assess changes in the genetic and epigenetic profiles of the target genes/genomes and identify potential molecular biomarkers for OC (21,53-56). KL is located at chromosome 13q12, with a length of 50 kb including a promoter region of 1,439 bp. It is a known tumor suppressor gene that has been reported to undergo aberrant promoter methylation in many tumors (35). The level of abnormal expression of KL in granulosa cells of the ovary is tightly linked with the acuteness of ovary-related diseases (37). Thus, in this study, the methylation pattern of KL were determined to evaluate its potential as a promising molecular biomarker in OC and assess its prognostic value. To the best of our knowledge, this is the first study conducted in the Arabian Peninsula to assess this biomarker in patients with OC and particular clinicopathological features. Unlike previous studies, most of our cohort was diagnosed with OC before menopause (66.1%) and was younger than 50 years of age (64.5%). In contrast, other studies reported OC as an age-related disease, and as mainly postmenopausal (57). Moreover, the incidence increased more noticeably in women over 65 years of age (58), and the median age at diagnosis was between 50 and 79 years (59,60). According to the data released by Cancer Research UK, 53% of British women diagnosed with OC were 65 years and older. Moreover, according to the American Cancer Society report, 50% of the diagnosed OC patients were aged 63 years or above (61). These findings highlight an interesting 15-year shift in the onset of OC in the Arabian Peninsula, and therefore, warrant additional studies to demystify such an early onset. Parity has been reported to reduce the risk of OC in contrast to nulliparous women (62). However, the majority of our patient cohort was parous women (69%), which may be attributed to the genomic blueprint, environmental factors, and/or lifestyle choices; however, this needs further investigation.
Till date, only Wiley et al have assessed the KL methylation profiles in 215 epithelial OC patients. They found that KL was hypermethylated in 14% of the OC patients compared with the controls and the hypermethylation status was associated with a significant reduction in DFS (46). Strikingly, approximately 62% of our cohort had hypermethylated KL, which is 4-fold higher than the results reported by Wiley et al (46). Our results showed significant associations between KL methylation profile and tumor subtype (P<0.0001). Different OC subtypes have been established with distinct molecular signatures (63,64). Our data showed KL promoter hypermethylation mainly in mucinous and serous subtypes. KL methylation showed borderline significance with age (P=0.064) (Table I). In contrast to the findings of Wiley et al (46), analysis of KL methylation profiles of OC patients did not reveal a significant correlation with DFS in our cohort (Fig. 2); however, a trend of faster recurrence in patients with KL promoter methylation was observed (Fig. 2). Moreover, DSS was significantly associated with KL methylation (P=0.03; log-rank test; Fig. 1). Our data showed that patients with higher KL promoter methylation were more likely to die earlier than those with non-methylated KL. In fact, in a 1-year follow-up, 75% of the patients with methylated KL were alive compared to those with non-methylated KL (100%). These results are in line with those of other tumors, mainly gastric, hepatocellular, and cervical carcinoma, where KL methylation profiles are associated with poor DFS (33,34,42). However, despite the association between downregulation of KL expression in follicular cells and ovarian diseases (37), the molecular mechanism of KL action and its relationship with aging and other diseases remains poorly understood and requires further investigation.
We acknowledge that our current OC study is a pilot study that gives clues about KL methylation prognosis value, and represents only the cohort of participants with possible inherent and unavoidable bias due to the size of the cohort and the available biological material and associated follow-up data. A large-scale multi-centric validation study is required to confirm these findings.
Our results showed that KL promoter hypermethylation is a poor prognosticator in OC patients. Patients with higher KL promoter methylation are more likely to die earlier, and therefore, more vigilant follow-up and integrative therapeutic approaches are required. Due to the increase in OC related mortality and the absence of early diagnosis or effective therapeutics, KL methylation could be used as an OC prognosticator to achieve better and personalized clinical management of this gynecological malignancy.