Homologous recombination inquiry through ovarian malignancy investigations: JGOG3025 Study

Abstract

The Cancer Genome Atlas (TCGA) network has clarified that ~50% of high-grade serous ovarian cancers show homologous recombination deficiency (HRD). However, the frequency of HRD in Japanese patients with ovarian cancer remains unclear. We aimed to identify the frequency of HR-associated gene mutations in Japanese patients with ovarian cancer. The JGOG3025 study is a multicenter collaborative prospective observational study involving 65 study sites throughout Japan. We recruited 996 patients who were clinically diagnosed with ovarian cancer before surgery from March 2017 to March 2019, and 701 patients were eligible according to the criteria. We used frozen tumor tissues to extract DNA and performed next-generation sequencing for 51 targeted genes (including 29 HR-associated genes) in 701 ovarian cancers (298 high-grade serous cases, 189 clear cell cases, 135 endometrioid cases, 12 mucinous cases, 3 low-grade serous cases, and 64 others). HRD was defined as positive when at least one HR-associated gene was mutated. The frequencies of HRD and tumor BRCA1/2 mutations were 45.2% (317/701) and 18.5% (130/701), respectively, in the full analysis set. Next, we performed multivariate Cox proportional hazards regression analysis for progression-free survival (PFS) and overall survival (OS). Advanced-stage ovarian cancer patients with HRD had adjusted hazard ratios of 0.72 (95% CI, 0.55–0.94) and 0.57 (95% CI, 0.38–0.86) for PFS and OS, respectively, compared with those without HRD (p = 0.016 and 0.007). Our study demonstrated that mutations in HR-associated genes were associated with prognosis. Further studies are needed to investigate the prognostic impact of each HR-associated gene in ovarian cancer.

Abbreviations
• aHR
• Adjusted hazard ratio
• CI
• Confidence interval
• FIGO
• International Federation of Gynecology and Obstetrics
• GI
• Genomic instability
• HR
• Homologous recombination
• HRD
• Homologous recombination deficiency
• JGOG
• Japanese Gynecologic Oncology Group
• MSI
• Microsatellite instability
• NAC
• Neoadjuvant chemotherapy
• OS
• Overall survival
• PARP
• Poly(ADP-ribose)polymerase
• PFS
• Progression-free survival
• PS
• Performance status
• SNV
• Single nucleotide variant
• TCGA
• The Cancer Genome Atlas
• ToMMo
• Tohoku Medical Megabank Organization
• VAF
• Variant allele frequency

INTRODUCTION

Homologous recombination deficiency has attracted attention as a new molecular biomarker in the gynecologic oncology field.¹ Homologous recombination repair is one of the repair mechanisms for DNA double-stranded breaks, in which the ends of double-stranded breaks are precisely repaired.² In humans, several genes, including BRCA1/2 and RAD51, are involved in HR repair, and genomic alterations in these HR-associated genes result in HRD. In HRD cells, DNA repair mechanisms other than HR repair are important, especially PARP, which is a new therapeutic target. PARP inhibition in HRD cells leads to genomic instability and cell death (synthetic lethality).³ At present, two PARP inhibitors (olaparib and niraparib) have become clinically available for ovarian cancer in Japan. Interestingly, two phase III trials have demonstrated that niraparib significantly prolonged PFS in not only patients with BRCA1/2 mutations, but also those with HRD.^4,5 The PAOLA-1 regimen (olaparib + bevacizumab) also provides a significant PFS benefit in patients with HRD.⁶ Therefore, HRD status is an important factor in determining the treatment of ovarian cancer.

Analysis based on TCGA, a national cancer genome project from the USA, revealed that approximately half of the high-grade serous ovarian cancers have genomic or epigenomic alterations in the HR-associated pathway, such as BRCA1/2 mutations, BRCA1 methylation, CDK12 mutations, and RAD51C promoter methylation.^7,8 However, the frequency of HR-associated mutations in ovarian cancer in Japan remains unclear, especially in non-serous histologic types. Because there are differences in the distribution of histologic types in ovarian cancer between the USA and Japan, the results of TCGA cannot be directly applied to Japan. In addition, the clinical significance of HRD remains unclear compared with that of germline BRCA1/2 mutations. Investigating the frequency and clinical significance of HRD in Japanese patients with ovarian cancer is an urgent issue.

Therefore, in 2017, we started the Japanese Gynecologic Oncology Group (JGOG) 3025 trial, which is a multicenter collaborative prospective observational study, to clarify the frequency of HRD in Japanese patients with ovarian cancer. We defined the presence of at least one HR-associated gene mutation as HRD in this study. We closed the enrollment of the participants at the end of March 2019 and fixed the data after 30 months of follow-up. We report the results of the final analysis of the JGOG3025 trial.

MATERIALS AND METHODS Patients and study design

A multicenter collaborative prospective observational study (ClinicalTrials.gov, NCT03159572; UMIN 000026303) was conducted in Japan between January 2017 and September 2021. We enrolled participants for 27 months between January 2017 and March 2019, and the follow-up period was 30 months after the end of enrollment. The target sample size was 700. The study schema is shown in Figure S1. Patients clinically diagnosed with ovarian cancer were enrolled from 65 JGOG institutions throughout Japan (Table S1). After written informed consent was obtained for the HRD test and biobanking, we collected tumor tissue samples during surgery. Tumor tissue samples were sent to the JGOG ToMMo biobank under controlled temperature. We performed an HRD test after the end of enrollment. When patients had information on germline BRCA1/2 mutations and allowed us to use the information in this study, germline BRCA1/2 mutation status was also submitted to the data center. Treatment selection was performed according to the Japan Society of Gynecologic Oncology guidelines for the treatment of ovarian cancer, fallopian tube cancer, and primary peritoneal cancer.^9,10 All evaluations were scheduled according to the clinical practice standards of each institution.

We collected data including patient characteristics, clinicopathological data, clinical outcomes, and adverse events. Clinical outcomes were updated prospectively every 6 months in the electronic data capture system.

The study was conducted in accordance with the Declaration of Helsinki and the Ethical Guidelines for Medical and Health Research Involving Human Subjects. Approval from the institutional review board of each participating JGOG institution was obtained prior to the initiation of the study. All patients provided written informed consent.

Study population

The inclusion criteria were as follows: patients who could approve informed consent and sign the form before surgery; patients who are clinically diagnosed with ovarian cancer; patients who are 20 years old and above at enrollment; patients with ECOG PS 0–2; and patients who could provide tumor tissue specimens. Ascites cytology and cell block specimens were not considered tumor tissue specimens. For patients to be treated with NAC, a tumor biopsy should be performed prior to NAC to make a pathologic diagnosis and obtain a tumor tissue specimen.

Patients were excluded based on the following criteria: patients with other clinically active malignancies (except breast cancer) that were treated within 5 years (excluding basal cell carcinoma and squamous cell carcinoma of the skin and carcinoma in situ or intramucosal carcinoma that are curable with local treatment); the principal investigator judged that enrollment of the patient in the study was inappropriate.

Study outcomes

The primary outcome of the JGOG3025 trial was to identify the HRD frequency in Japanese patients with ovarian cancer, including fallopian tube cancer and primary peritoneal cancer. The secondary outcomes were associations between PFS/OS and HRD in ovarian cancer and between PFS/OS and germline BRCA1/2 mutation in ovarian cancer. PFS was defined as the time from registration in the trial to disease progression or death from any cause.

Definition of HRD

We performed next-generation sequencing for 51 targeted genes using DNA extracted from cancer tissues. A list of the genes is shown in Table S2. Five genes (ARID1A, KRAS, PIK3CA, PTEN, and TP53) were included as molecular diagnostic markers in ovarian cancer, and 29 genes (ATM, ATR, ATRX, BAP1, BARD1, BLM, BRCA1, BRCA2, BRIP1, CHEK1, CHEK2, EMSY, FANCA, FANCC, FANCL, MLE11, NBN, PALB2, POLD1, RAD21, RAD50, RAD51, RAD51B, RAD51C, RAD51D, RAD52, RAD54L, RECQL4, XRCC2) were categorized as HR-associated genes. We used a QIAseq Targeted DNA Custom Panel (CDHS-16649Z-2451, Qiagen) and conducted 150-bp paired-end sequencing on an Illumina NextSeq sequencer. Single nucleotide variants or short InDels were detected using smCounter2 (Qiagen).¹¹ Variant annotation was performed using ANOVAR version 20190408.¹²

To make a mutation list, we generated a mutant allele frequency (MAF) file using maftools¹³ and filtered it as follows: variants with VAF less than 5% were excluded; variants classified as Nonsense, Frame Shift InDel, Nonstop, Translation Start Site, Missense, and Splice Site were included; variants with a VAF of more than 1% in gnomAD,¹⁴ 1000 Genomes Project,¹⁵ ExAC,¹⁶ ESP6500,¹⁷ or 14 K Japanese custom reference from jMorp¹⁸ were excluded; variants interpreted as pathogenic or are likely to be pathogenic in ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/) or InterVar¹⁹ were included; based on dbsnpf35a or dbscsnv11, missense variants with deleterious 2 predictions or splice variants with deleterious >1 prediction were included; and variants that were found in neither COSMIC²⁰ nor ICGC²¹ were excluded.

For HRD, we defined cases harboring at least one mutation of HR-associated genes as HRD and cases without any HR-associated gene mutation as non-HRD. Because MSI-high or POLE-mutated ovarian cancers showing a very high tumor mutational burden led to passenger mutations of HR-associated genes, cases with both HR-associated gene mutations and MSI-high or POLE mutations were determined to be HRD negative.

Central pathological review

To confirm the histological diagnosis, the central pathological review was performed by three pathologists (Professor Yuko Sasajima, Professor Miki Kushima, and Dr. Masaharu Fukunaga) assigned by the JGOG using H&E-stained slide specimens from tumor tissues. The central histological evaluation and diagnosis of ovarian cancer were based on the WHO classification of tumors of female reproductive organs.

Statistical analysis

The analysis population was the full analysis set of all ovarian cancer patients for whom sample tumor tissue was submitted and who met study enrollment criteria, based on the intention-to-treat (ITT) principle. The frequency of HRD and the primary outcome were calculated as a percentage and 95% CI. The cumulative survival rates for PFS and OS were estimated using the Kaplan–Meier method, and the incidence rate and its 95% CI were calculated. Patients who discontinued the study without having an event were analyzed as censored cases at the time of discontinuation. Adjusted hazard ratios for HRD positive/HRD negative and its 95% CIs were calculated using a stratified proportional hazards model with age, stage, histologic type, surgery, NAC, and adjuvant chemotherapy as covariates, stratified using the FIGO classification of stage I/II and stage III/IV. Imputation of missing values was not performed for either the primary or secondary outcomes. Data are expressed as mean (SD) for continuous variables, and frequencies and percentages for discrete variables, unless specifically mentioned. The p-values were two-sided, and p-values less than 0.05 were considered significant. The data were analyzed using SAS version 9.4 (SAS Institute, Inc., Cary, NC, USA).

RESULTS Patient characteristics

The study was conducted from June 2015 to November 2019. The enrollment period was initially planned for 2 years but was extended to 2 years and 3 months to reach the sample size needed. The patient disposition is shown in Figure 1. In total, 996 patients who were clinically diagnosed with ovarian cancer were enrolled, but 295 patients were excluded from the full analysis set according to the eligibility criteria. In total, 701 patients were included in the final analysis. The patient characteristics are shown in Table 1. Patients in the full analysis set had a mean age of 58.4 years. Of 701 patients, 47.4% had stage I/II disease, and 51.6% had stage III/IV disease. The mean time from registration to the last follow-up was 2.82 ± 0.87 years in the full analysis set.

FIGURE 1. Patients flow chart.

TABLE 1 Clinicopathological characteristics of FAS

	FAS (n = 701)
Age
<65 years	474 (67.6%)
≥65 years	227 (32.4%)
FIGO stage
I/II	332 (47.4%)
III/IV	362 (51.6%)
Unknown	7 (1.0%)
Histologic type
High-grade serous carcinoma	298 (42.5%)
Clear cell carcinoma	189 (27.0%)
Endometrioid carcinoma	135 (19.3%)
Mucinous carcinoma	12 (1.7%)
Low-grade serous carcinoma	3 (0.4%)
Others	64 (9.1%)
Surgery
Complete	520 (74.2%)
Optimal (<1 cm)	75 (10.7%)
Not optimal (≥1 cm)	104 (14.8%)
Unknown	2 (0.3%)
Neoadjuvant chemotherapy
Yes	152 (21.7%)
No	549 (78.3%)
Adjuvant chemotherapy
Yes	612 (87.3%)
No	89 (12.7%)

Primary outcome

The mutation profiles of HR-associated genes per sample are shown in Figure 2A. After cases with both HR-associated gene mutations and MSI-high or POLE mutations were determined to be HRD negative, the frequency of HRD in Japanese patients with ovarian cancer was 45.2% (317/701) (Figure 2B). The mutation rates of tumor BRCA1 and BRCA2 were 9.8% and 7.7%, respectively. Seven patients (1.0%) had both BRCA1 and BRCA2 mutations. Conversely, the mutation rate of HR-associated genes except BRCA1/2 was 26.7%.

FIGURE 2. The frequency of HRD in Japanese patients with ovarian cancer. (A) The heatmap demonstrates the mutation status of 29 HR-associated genes per sample. (B) The pie chart shows the frequency of BRCA1, BRCA2, and other HR-associated gene mutations.

Secondary outcomes

First, we performed a Cox proportional hazards model analysis after adjustment for age, stage, histologic type, surgery, NAC, and adjuvant chemotherapy. There was no significant difference in PFS between the HRD and non-HRD groups (aHR, 0.85; 95% CI, 0.67–1.07; p = 0.17) (Figure S2A). Conversely, the HRD group had a longer OS than the non-HRD group (aHR, 0.68; 95% CI, 0.47–1.00; p = 0.049) (Figure S2B).

Next, we stratified ovarian cancers by FIGO stage. Although there was no significant difference in PFS between the stage I/II HRD and non-HRD subgroups (aHR, 1.36; 95% CI, 0.82–2.25; p = 0.24), the stage III/IV HRD subgroup had a significantly longer PFS than the non-HRD subgroup (aHR, 0.72; 95% CI, 0.55–0.94; p = 0.016) (Figure 3). Similarly, there was no significant difference in OS between the stage I/II HRD and non-HRD subgroups, but the stage III/IV HRD subgroup had a significantly longer OS than the non-HRD subgroup (aHR, 0.57; 95% CI, 0.38–0.86; p = 0.007) (Figure 4).

FIGURE 3. Association between PFS and HRD in stage I/II (A) and stage III/IV (B) ovarian cancer.

FIGURE 4. Association between OS and HRD in stage I/II (A) and stage III/IV (B) ovarian cancer.

The frequency of HRD by histologic type is shown in Table 2. In patients with high-grade serous carcinoma, the frequencies of both HRD and tumor BRCA1/2 mutations were relatively higher than those in other histologic types. Although the frequency of HRD in the clear cell type was also relatively high, the tumor BRCA1/2 mutation rate was lower than that in high-grade serous carcinoma. When we focused on both stage and histologic type, there were no significant differences in PFS and OS between the HRD and non-HRD subgroups of stage III/IV high-grade serous carcinomas (Figure S3). Conversely, the HRD subgroup of stage III/IV clear cell carcinomas had a better prognosis than the non-HRD subgroup (Figure 5).

TABLE 2 The frequency of HRD by histologic type

Histology	N	HRD	BRCA1	BRCA2	BRCA1 + 2
High-grade serous carcinoma	298	154 (51.7%)	52 (17.4%)	33 (11.1%)	2 (0.7%)
Clear cell carcinoma	189	81 (42.9%)	6 (3.2%)	2 (1.1%)	0 (0%)
Endometrioid carcinoma	135	49 (36.3%)	6 (4.4%)	16 (11.9%)	3 (2.2%)
Mucinous carcinoma	12	3 (25.0%)	0 (0%)	0 (0%)	0 (0%)
Low-grade serous carcinoma	3	3 (100%)	0 (0%)	0 (0%)	0 (0%)
Others	64	27 (42.2%)	5 (7.8%)	3 (4.7%)	2 (3.1%)

FIGURE 5. Association between PFS (A) /OS (B) and HRD in stage III/IV clear cell carcinomas.

Then, we focused on germline BRCA1/2 mutation status in ovarian cancer. We collected germline BRCA1/2 information from 112 participants. Of them, germline BRCA1/2 mutations were positive in 22 participants (19.6%). Nineteen of these were identified in the corresponding tumors in this study cohort. Conversely, five participants with tumor BRCA1/2 mutations did not have germline BRCA1/2 mutations. There was no significant difference between PFS and germline BRCA1/2 mutations in stage I/II ovarian cancer because only three participants harbored germline BRCA1/2 mutations (aHR, 1.14; 95% CI, 0.08–16.7; p = 0.92). By contrast, germline BRCA1/2-mutated stage III/IV ovarian cancers showed a significantly longer PFS than germline BRCA1/2 wild-type stage III/IV ovarian cancers (aHR, 0.28; 95% CI, 0.12–0.65; p = 0.003) (Figure 6). In addition, germline BRCA1/2 mutation was significantly associated with OS in stage III/IV ovarian cancer (aHR, 0.18; 95% CI, 0.05–0.70; p = 0.013).

FIGURE 6. Association between germline BRCA1/2 mutation with PFS (A) and OS (B) in stage III/IV ovarian cancer.

Mutant allele frequency (MAF) of HR-associated genes

To examine the impact of HR-associated gene mutations in ovarian cancer, we measured the MAF per each gene (Figure S4). Intriguingly, the median of BRCA1 MAF was 0.80, suggesting that most ovarian cancer with BRCA1 mutation occurred in the loss of function of BRCA1. BRCA2, RAD51D, and BAP1 also showed high MAF (>0.5) in a relatively large number of cases. Conversely, the MAF peaks of other HR-associated genes were located at ~0.4–0.5.

DISCUSSION

Our multicenter collaborative prospective observational study revealed that the frequency of HRD in Japanese patients with ovarian cancer was 45.2% and that HRD was significantly associated with PFS and OS in advanced-stage ovarian cancer.

In TCGA dataset, 40.5% of high-grade serous ovarian cancers had genomic or epigenomic alterations in the HR-associated pathway.^7,8 In the PAOLA-1 trial, HR status was assessed using the myChoice HRD PLUS assay (Myriad Genetic Laboratories, Inc.), and 48.0% of high-grade serous or endometrioid carcinomas were diagnosed as HRD.⁶ The first real-world data analysis from China demonstrated that 46/67 ovarian cancer patients were diagnosed as HRD positive based on the AmoyDx® HRD panel that measures BRCA status and HRD score.²² Pujade-Lauraine et al. have also reported the 13 HR-associated gene mutation status in the exploratory analysis of the PAOLA-1 trial (DOI: https://doi.org/10.1016/S0090-8258(21)00695-8). Of 806 cases in the PAOLA-1 trial, 235 (29.2%) had a tumor BRCA mutation. HR-associated gene mutations (excluding tBRCA) were also detected in 54 cases (6.7%). PanCancer Atlas data from cBioPortal (https://www.cbioportal.org)²³ showed that 346/523 high-grade serous ovarian carcinomas (66%) had at least one mutation in 29 HR-associated genes that were analyzed in this study. Our result was relatively consistent with those of previous studies, suggesting that HRD was present in a certain percentage of ovarian cancers across ethnic groups.

HRD was significantly associated with prognosis in stage III/IV ovarian cancer but not in stage I/II ovarian cancer. The distribution of histologic types differed between stage I/II and stage III/IV, and the proportion of high-grade serous carcinoma cases was higher in stage III/IV than in stage I/II. Therefore, the frequency of BRCA1/2 mutations was also higher in stage III/IV. It is well known that BRCA1/2 mutation is associated with platinum sensitivity in ovarian cancer,²⁴ and BRCA1/2 status might affect the present results. Conversely, we also found associations between PFS/OS and HRD in stage III/IV clear cell carcinoma, which harbored fewer BRCA1/2 mutations. These results suggested that other HR-associated gene mutations are linked with platinum sensitivity in advanced-stage clear cell carcinoma.²⁵ Intriguingly, our study showed that not only high-grade serous histologic type, but also clear cell or mucinous histologic types, have a high percentage of HRD positivity as we assessed HR-associated gene mutation status. Some clear cell or mucinous carcinoma cases with HR-associated gene mutation might benefit from PARP inhibitors.

Olaparib maintenance therapy for platinum-sensitive relapsed ovarian cancer was approved by health insurance in January 2018 in Japan. The SOLO2 trial showed that the olaparib treatment group had a longer OS (median 51.7 months; 95% CI 41.5–59.1) than the placebo group (median 38.8 months; 95% CI 31.4–48.6) (HR 0.74; 95% CI 0.54–1.00; p = 0.054), although 38% of the placebo group received subsequent PARP inhibitor therapy.²⁶ In addition, niraparib for platinum-sensitive relapsed or HRD ovarian cancers was also approved by health insurance in September 2020. Therefore, it is necessary to verify whether there is a difference in the rate of PARP inhibitor administration between the HRD and non-HRD groups.

In this study, HRD was defined as the presence of at least one HR-related gene mutation. In clinical practice, the Myriad myChoice diagnostic system is now covered by health insurance in Japan and is the standard method for assessing GI scores calculated by genomic loss of heterozygosity (LOH),²⁷ telomeric allelic imbalance (TAI),²⁸ and large-scale state transition (LST).²⁹ Recently, the assessment of HRD based on mutational signatures has also been proposed.^30,31 However, the two methods reflect the genomic scar causing HRD and do not indicate whether the current tumor is HRD. Conversely, HR-associated gene mutation is one of the etiologies of HRD. In other words, BRCA1/2 mutation status is the only commonality between our method and the Myriad myChoice diagnostic system. BRCA1/2 mutations are significantly correlated with the GI score and significantly associated with clinical outcomes in ovarian cancer. However, the clinical significance of HR-associated genes other than BRCA1/2 remains unclear. Recently, subsequent PAOLA-1 analysis demonstrated that HR-associated gene mutation (excluding BRCA1/2 mutation) was not predictive of PFS benefit with maintenance olaparib in combination with bevacizumab compared with bevacizumab alone in the PAOLA-1 trial and that only five genes (BLM, BRIP1, RAD51C, PALB2, and RAD51D) had a median GI score ≥ 42.³² Conversely, Loverix et al. have demonstrated that the Leuven HRD test has a robust correlation with Myriad myChoice PLUS test (https://doi.org/10.1016/S0090-8258(22)01299-9). The correlation between our method and the Myriad myChoice diagnostic system should be investigated in terms of the predictive value of PARP inhibitors. Furthermore, multiomics data analysis is needed to determine the clinical impact of each HR-associated gene mutation in ovarian cancer.

Our study has several limitations. The HR-associated mutations may be monoallelic, without any alterations in the opposite allele. Although tumors with MSI-high or POLE mutations were excluded from HRD, other HR-associated mutations may also not contribute to tumorigenesis. As the correlation between the GI score and HR-associated mutations has not been analyzed, the HRD status in each tumor has not been fully verified. In addition, target sequencing cannot identify tumors with epigenetic alterations (such as hypermethylation in the promoter region in BRCA1 or RAD51C). In addition, we enrolled patients who were clinically diagnosed with ovarian cancer and could provide tumor tissue samples after surgery. Compared with the real-world distribution of each histology based on the Japan Society of Obstetrics and Gynecology (JSOG) annual report,³³ the proportions of mucinous carcinoma and low-grade serous carcinoma were smaller in the JGOG3025 trial. This may be due to the difficulty of preoperative diagnosis and tumor tissue sampling in mucinous and low-grade serous carcinomas.

In conclusion, HRD was detected in not only high-grade serous histologic type but also other histologic types, and HRD status may be useful in providing an effective treatment option for patients with ovarian cancer.

ACKNOWLEDGMENTS

We wish to thank all members of the JGOG. We appreciate all the women who participated in this study and their families, the staff of the Translational Research Center for Medical Innovation (TRI; the data and statistical analysis center for JGOG 3025), and the participating JGOG member institutions.

FUNDING INFORMATION

This study was partially funded by AstraZeneca Externally Sponsored Research.

CONFLICT OF INTEREST STATEMENT

The authors except for Kosuke Yoshihara, Tsukasa Baba, Noriomi Matsumura, Katsutoshi Oda, Aikou Okamoto, and Takayuki Enomoto declare that they have no conflicts of interest. Kosuke Yoshihara received lecture fees and a research grant from AstraZeneca. Tsukasa Baba received lecture fees from AstraZeneca and Chugai. Noriomi Matsumura received lecture fees from AstraZeneca and Takeda Pharmaceuticals. Noriomi Matsumura also received a research grant from AstraZeneca. Katsutoshi Oda received lecture fees from AstraZeneca, Chugai, and Takeda Pharmaceuticals. Katsutoshi Oda also received a research grant from AstraZeneca. Aikou Okamoto received lecture fees from AstraZeneca, Chugai, MSD, Mochida, Bayer, Kaken, ASKA, Kissei, Fuji, and Takeda Pharmaceuticals. TE received lecture fees from AstraZeneca, Chugai, MSD, and Takeda Pharmaceuticals. Katsutoshi Oda is an editorial board member of Cancer Science.

ETHICS STATEMENT

Approval of the research protocol by an institutional review board:This study was approved by the institutional review board of each participating JGOG institution.

Informed Consent: All patients provided written informed consent.

Registry and Registration No. of the study/trial: NCT03159572 (ClinicalTrials.gov); UMIN 000026303 (UMIN-CTR).

Animal Studies: N/A.