{"paper_id":"f948ee21-ecbd-4b76-972f-854cdf8ab05c","body_text":"Ovarian cancer remains the cancer with the worst survival rates in women, as in most cases it is diagnosed at an advanced stage [ 1 ]. It is the second most frequent malignancy, following breast cancer, in women over the age of 40, especially in developed countries [ 2 ]. For primary disease, the customary treatment is debulking surgery accompanied by first-line platinum and paclitaxel-based chemotherapy [ 1 ]. The majority of patients respond to primary treatment but more than half of them will acquire chemo-resistance and consequently recurrent disease [ 1 ]. Epithelial ovarian cancer is the most frequent type, with histological and molecular heterogeneity [ 1 , 2 ]. Serous tumors are classified into high-grade serous carcinomas (HGSCs) and low-grade serous carcinomas (LGSCs) [ 2 ]. The former is a highly aggressive disease that is often diagnosed at an advanced FIGO (International Federation of Gynecology and Obstetrics) stage [ 1 ]. Overall survival (OS) remains low, even though there have been slight improvements in therapy [ 1 ]. There are a few available targeted therapies for ovarian cancer, such us the anti-angiogenetic antibody bevacizumab [ 3 ] and the PARP (poly (ADP-ribose) polymerase) inhibitors olaparib and rucaparib, which are FDA (Food and Drug Administration)-approved for platinum-sensitive recurrent BRCA-mutated ovarian cancer patients [ 4 , 5 ].\nThe most commonly reported gene mutations, highly associated with epithelial ovarian cancer, are for  TP53 ,  BRCA1/2 ,  PIK3CA  and  KRAS  genes [ 6 ]. The frequency of the above-mentioned mutations varies among the subtypes of epithelial ovarian cancer. Mutations in  TP53  are present in more than 96% of ovarian cancer cases [ 6 , 7 ].  BRCA1/2  mutations are associated with the majority of hereditary ovarian cancer or Lynch syndrome and the mutation rate of  BRCA1/2  increases in recurrent HGSOC [ 6 , 7 ].  PIK3CA  mutations have been also detected at high frequencies in ovarian clear cell carcinoma (OCCC) and endometrioid ovarian cancer related to endometriosis [ 6 ].  NOTCH3  mutations have been detected in 66% of HGSOC cases and  NOTCH3  inactivation could be a potential therapeutic approach [ 7 ]. Low-grade serous ovarian carcinomas (LGSOCs) are associated with  BRAF  (especially V600E) and  KRAS  mutations [ 6 , 7 , 8 ]. LGSOCs present with a lower frequency of somatic  TP53  and  BRCA1/2  mutations and are not associated with germline  BRCA1/2  mutations [ 8 ].  RAD51C  and  RAD51D  have been demonstrated to be inherited ovarian cancer predisposition genes with mutation carriers showing HGSOC [ 8 ]. In addition, the deleterious germline mutations BRP1 (BRCA1-interacting protein 1) are mainly associated with the high-grade serous epithelial subtype [ 8 , 9 ].\nLiquid biopsy, now widely recognized as an important tool for the follow-up of cancer patients, is mainly based on the analysis of circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA), which provide a source of diagnostic or/and prognostic markers. The clinical significance of CTCs and ctDNA in ovarian cancer has been investigated in many studies to date [ 1 , 10 , 11 , 12 , 13 , 14 , 15 , 16 ]. We have recently reported that  ESR1  is methylated in HGSOC patients, and that there is a statistically significant concordance between  ESR1  methylation in primary tumors and paired ctDNA [ 17 ]. Recently, many studies have investigated the mutation profile of ovarian cancer patients in plasma-cfDNA in many genes, such as  TP53 ,  PIK3CA ,  KRAS ,  BRAC1 ,  BRAC2  and  EGFR  [ 18 , 19 , 20 , 21 , 22 , 23 ].\nESR1  mutations have emerged as a key mechanism of resistance to endocrine therapy in patients with ER-positive metastatic breast cancer [ 24 ] and their detection is now considered to be highly promising as a prognostic and predictive biomarker in this type of cancer [ 24 , 25 ]. To date, only a few studies have investigated the presence of  ESR1  mutations in endometrial and cervical cancer [ 26 , 27 , 28 , 29 , 30 ]. According to the cBioPortal cancer genomics database,  ESR1  mutations have been detected in 4–6% of uterine corpus endometrial carcinoma samples [ 31 ]. As for ovarian cancer, the cBioPortal cancer genomics database includes one ovarian serous cystadenocarcinoma study in which  ESR1  mutations were detected in 0.8% of samples [ 31 ]. In 2018, Stover et al., using targeted next-generation sequencing (NGS), detected a Y537S  ESR1  mutation in one patient with low-grade serous ovarian cancer (LGSOC); this particular patient developed a single site of progressive disease in an abdominal wall nodule and maintained stable low-volume peritoneal disease during endocrine therapy for almost five years, but later presented progressive disease after a durable response to hormonal therapy [ 32 ].\nThe aim of our study was to evaluate the frequency and the clinical relevance of  ESR1  mutations in HGSOC (HGSOC). We applied our recently developed highly sensitive and specific ESR1-NAPA assay for the detection of  ESR1  hotspot mutations (Y537S, Y537C, L536R, Y537N and D538G) [ 33 ] in combination with drop-off ddPCR [ 34 ] to investigate  ESR1  mutational status in primary tumors and plasma cfDNA in HGSOC patients.\n\nThe study material consisted of (a) primary formalin-fixed paraffin-embedded tumor tissues (FFPEs) from patients with HGSOC prior to any systemic treatment ( n  = 60) and, as a corresponding non-cancerous control, a group of 10 normal fallopian tube FFPEs that were obtained from women at the productive age; and (b) plasma-cfDNA samples from 80 patients with advanced ( n  = 20) and metastatic ovarian cancer ( n  = 60), and as a corresponding control, plasma-cfDNA samples from female healthy donors (HD,  n  = 11). All patients received at least six cycles of carboplatinum AUC 5 and paclitaxel at 175 mg/m 2 . Patients provided written informed consent to participate in the study, which was approved by the Local Essen Research Ethics Committee (16-6916-BO; 17-7859-BO), and the General University Hospital of Alexandroupolis’ ethics committee (date: 25 June 2020). The available clinicopathological features are shown in  Table 1  and  Table 2 .\nFFPEs: FFPEs containing >60% tumor cells were used for genomic DNA (gDNA) extraction. gDNA was isolated from FFPEs with using the QIAamp ®  DNA FFPE Tissue Kit 50 (Qiagen ® , Hilden, Germany), according to the manufacturer’s instructions. The DNA concentration was determined using a Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, NC, USA).\nPlasma: 10 mL of peripheral blood in EDTA were used within 2–4 h to isolate plasma via centrifugation at 530×  g  for 10 min. Following a second centrifugation at 2000×  g  for 10 min, plasma was transferred into 2 mL tubes and stored at −70 °C until use. cfDNA was further isolated using the QIAamp Circulating Nucleic Acid Kit (Qiagen, Hilden, Germany), as previously described [ 35 ].\nIn all samples, cfDNA quality was checked prior to PCR using a previously described protocol [ 36 ]. Serial dilutions of a wild-type sample with a known DNA concentration (Human Reference DNA Female, Agilent Technologies, Santa Clara, CA, USA), prepared via serial 10-fold dilution in concentrations ranging from 200 ng/μL down to 0.5 ng/μL, were used to generate a standard curve for the quantification of the gDNA concentration in all cfDNA samples using a LightCycler z480 (Roche).\nAll cfDNA samples and controls were screened for  ESR1  mutations in exon 8, including the Y537S, Y537C, Y537N, D538G and L536R mutations, using drop-off ddPCR in a QX200 Droplet Digital PCR System (Bio-Rad Laboratories, Hercules, CA, USA), as previously described [ 34 ].\nAll samples that were found to be positive for  ESR1  mutations via the  ESR1  drop-off ddPCR and all controls were further analyzed to define each individual  ESR1  mutation using our previously developed and validated ultrasensitive  ESR1 -NAPA assay for Y537S, Y537C, Y537N and D538G mutations [ 33 ]. Synthetic oligonucleotide sequences for each individual  ESR1  mutation were used as positive controls. In this study, we additionally designed, analytically validated and added the L536R mutation into our  ESR1 -NAPA assay. The experimental conditions for the  ESR1 -L536R mutation assay were optimized in detail regarding the annealing temperature, time and concentration of primers, buffer, MgCl 2  (magnesium chloride solution), dNTPs (deoxyribonucleotide triphosphates) and BSA (bovine serum albumin solution) (data not shown).\nSPSS version 28.0 (IBM ®  SPSS ®  Statistics, Endicott, NK, USA) was used for statistical analysis. Pearson’s χ 2  and Cohen’s kappa coefficient tests were used to estimate the concordance between  ESR1  mutations in primary tumors and paired cfDNA. The correlation between  ESR1  mutations and the clinicopathological characteristics of the patients ( Table 1 ) were estimated using Pearson’s χ 2  and Fischer’s exact test ( p -values < 0.05 were considered statistically significant). Kaplan–Meier analysis was used for overall survival (OS) and progression-free survival (PFS) curves.\n\nA schematic flowchart of our study is given in  Figure 1 .\nTo ensure the specificity of the drop-off ddPCR assay we first evaluated the mutant allelic frequency (MAF) in 10 non-cancerous fallopian tube samples. A cut-off value was calculated by adding the 2SD (standard deviation) to the mean of the MAF values of these control samples. The MAF% was estimated using the program developed by Attali et al. specifically for this type of ddPCR assay [ 37 ]. Based on the defined cut-off (1.15), we detected the presence of  ESR1  mutations in 9/60 (15%) of FFPE samples tested ( Figure 2 ).\nIn this patient group the median PFS was 41 months, and the median OS was 47 months. There was no significant correlation between OS, PFS, and  ESR1  mutations in FFPEs when our results were evaluated via Kaplan–Meier analysis (data not shown). Furthermore, no significant correlation between  ESR1  mutations and the patients’ clinicopathological characteristics was observed.\nUsing drop-off ddPCR,  ESR1  mutations were detected in 11/80 (13.8%) plasma-cfDNA samples ( Figure 3 ), more specifically, in eight plasma-cfDNA samples from patients with metastatic ovarian cancer (8/60, 13.3%) and in three plasma-cfDNA samples from patients with advanced ovarian cancer (3/20, 15%). All these  ESR1 -mutation-positive samples were further analyzed to define  ESR1  mutations using the  ESR1 -NAPA assay ( Figure 4 ). The D538G mutation was detected in three plasma-cfDNA samples from patients with metastatic ovarian cancer and L536R was detected in two plasma-cfDNA samples from patients with metastatic ovarian cancer and in one plasma-cfDNA sample from one patient with advanced ovarian cancer. It should be mentioned that in one patient with metastatic ovarian cancer, both D538G and L536R were detected in plasma-cfDNA.\nThe median PFS was 38 months and the median OS was 38 months in the group of  n  = 20 patients with advanced ovarian cancer, and the median PFS was 19 months and the median OS was 31 months in the group of  n  = 60 patients with metastatic ovarian cancer. Kaplan–Meier analysis was performed to estimate the correlation between OS and PFS with the detection of  ESR1  mutations in both groups. No significant correlations were observed among OS, PFS and  ESR1  mutations for both groups (data not shown). Furthermore, no significant correlations between  ESR1  mutations and the patients’ clinicopathological characteristics were observed.\n\nWe report, for the first time, the detection of  ESR1  mutations in primary tumors (FFPEs) in plasma cfDNA samples from patients with advanced and metastatic ovarian cancer patients using highly sensitive and specific methodologies based on drop-off ddPCR for screening and the  ESR1 -NAPA assay for the definition of Y537S, Y537C, Y537N, L536R and D538G  ESR1  mutations.\nTo date, the detection of  ESR1  mutations has been reported in cervical squamous cell carcinoma [ 26 ] and in a patient with endometrial cancer treated with an aromatase inhibitor [ 27 ]. It has also been reported that the presence of  ESR1  mutations is associated with worse outcomes in endometrial cancer [ 28 ]. Apart from endometrial cancer studies, there are very few studies that show the existence of  ESR1  mutations in ovarian cancer. More specifically, in 2017, McIntyre et al. detected  ESR1  Y537S mutation in one patient with low-grade serous ovarian carcinoma, when analyzing 26 primary tumor samples using NGS [ 38 ]. In 2018, Stover et al., using targeted NGS, detected a Y537S  ESR1  mutation in one patient with LGSOC; this particular patient developed a single site of progressive disease in an abdominal wall nodule and maintained stable low-volume peritoneal disease during endocrine therapy for almost five years, but later presented progressive disease after a durable response to hormonal therapy [ 32 ]. In 2019, Gaillard et al. reported that  ESR1  mutations were detected in 4.4% (24/548) of uterine endometrioid carcinomas vs. 0.2% (1/446) of uterine serous carcinomas and 3.5% (5/144) of ovarian endometrioid carcinomas compared to 0.3% (12/3502) of ovarian serous carcinomas, whereas in an ovarian serous carcinoma both  ESR1  Y537S and D538G mutations were detected [ 39 ]. Since then, there have been no reports on the detection of  ESR1  mutations in ovarian cancer.\nIn the present study, we report that, using drop-off ddPCR,  ESR1  mutations were detected in 15% of primary tumor tissues and in 13.8% of plasma-cfDNA samples tested. More specifically, eight plasma-cfDNA samples from patients with metastatic cancer and three plasma-cfDNA samples from patients with advanced ovarian cancer were found to be positive for  ESR1  mutations. All plasma-cfDNA samples found to be positive via  ESR1  drop-off ddPCR were further analyzed using the  ESR1 -NAPA assay in order to define the specific mutation. In patients with metastatic ovarian cancer, the D538G mutation was detected in three plasma-cfDNA samples and L536R was detected in two plasma-cfDNA samples, whereas both D538G and L536R were detected in one patient. In patients with advanced ovarian cancer, L536R was detected only in one plasma-cfDNA sample. Drop-off ddPCR screens for  ESR1  mutations were clustered in exon 8. Hence, any mutation in this region could be detected in addition to Y537S, Y537C, Y537N, L536R and D538G. In this region, additional mutations were present, such as L536H, which has been detected in endometrial cancer [ 29 , 39 ].\nThese findings could be of clinical importance if we consider that in metastatic breast cancer the detection of  ESR1  mutations has led to the development of novel highly promising therapeutic strategies. In patients with metastatic breast cancer that are positive for  ESR1  mutations, selective estrogen receptor modulators (SERMs) and selective estrogen receptor covalent antagonists (SERCAs) are now being evaluated as promising drugs. Lasofoxifene is currently in Phase 2 trials for patients with  ESR1  mutations and for patients after progression on endocrine therapy and CDK4/6 inhibition [ 40 ]. The FDA has granted a fast-track designation to lasofoxifene for use as a treatment of female patients with estrogen receptor (ER)-positive, HER2-negative metastatic breast cancer who harbor  ESR1  mutations. Bazedoxifene, a SERM/SERD hybrid, which has been approved for use in postmenopausal hot flashes and osteoporosis, is now in a Phase 2 trial for patients after progression on endocrine therapy ( NCT02448771 ) [ 40 ]. Fanning et al. reported that bazedoxifene possessed improved inhibitory potency against the Y537S and D538G mutants compared to tamoxifen and had additional inhibitory activity in combination with the CDK4/6 inhibitor palbociclib [ 41 ]. In parallel, the efficacy of H3B-6545, a drug optimized from the SERCA class, against  ESR1  mutations was demonstrated in patients with metastatic breast cancer previously treated with endocrine therapy and CDK4/6i [ 42 ]. H3B-6545 is now in a Phase 2 trial for patients after progression on endocrine therapy and CDK4/6i ( NCT03250676 ) [ 40 ]. The combined analysis of SoFEA and EFFECT showed that patients with  ESR1  mutations detected in plasma-cfDNA samples [ 43 ] had shorter PFS and OS when treated with exemestane therapy, compared with fulvestrant. In the PALOMA-3 trial, patients on fulvestrant and a placebo tended to have poorer PFS in the presence of mutations compared to the absence of mutations [ 44 ]. O’Leary et al. reported that  ESR1  Y537S mutation promotes resistance to fulvestrant and that acquired mutations from fulvestrant are a major driver of resistance to fulvestrant and palbociclib combination therapy [ 45 ]. In the phase 3 PADA-1 trial presented at the 2021 San Antonio Breast Cancer Symposium, it was observed that when switching from an aromatase inhibitor plus palbociclib to fulvestrant and palbociclib upon early identification of the  ESR1  mutation in plasma—before disease progression—the median PFS was doubled. This trial has also shown that  ESR1  mutations are rarely detected in the plasma-cfDNA of ER + HER2− metastatic breast cancer patients with no overt resistance to aromatase inhibitors and that the detection of  ESR1  mutations was associated with a significantly shorter PFS, suggesting that the presence of the  ESR1  mutation at baseline could accelerate the outset of resistance to AI-palbociclib [ 46 , 47 ]. Novel therapies could include possible strategies to overcome the endocrine resistance induced by  ESR1  mutations.\n\nTo our knowledge, this is the first time that the presence of  ESR1  mutations has been reported in primary tumors and plasma-cfDNA from HGSOC patients. The clinical significance of this finding should be examined prospectively in a large group of ovarian cancer patients.","source_license":"CC-BY-4.0","license_restricted":false}