{"paper_id":"c8a0a3a1-b62d-446c-996b-c54f75b6f006","body_text":"The AT-rich interaction domain 1A ( ARID1A ) gene is located on chromosome 1p36.11 and encodes ARID1A ,  a key component of the SWI/SNF complex 1 . The SWI/SNF complex plays an important role in chromatin remodeling and is associated with numerous biological functions, such as differentiation and proliferation 2 . Therefore, aberrations in the SWI/SNF complex subunits have the potential to cause cancer. In particular,  ARID1A , which is well known as a tumor suppressor gene, is frequently mutated in a wide variety of cancers 3 , 4 . COSMIC data demonstrated that more than half of  ARID1A  mutations are loss-of-function mutations, including frameshift indels mutations, and nonsense mutations, that lead to a loss of ARID1A protein expression in cancer cells 5 .\nARID1A  mutation is considered one of the most important driver events in endometriosis-associated ovarian cancer 6 – 8 . According to previous studies, including ours 6 , 7 , 9 – 13 , 46–70% of clear cell carcinomas and 30–46% of endometrioid carcinomas harbor  ARID1A  mutations, and immunohistochemical analysis has demonstrated that  ARID1A  loss-of-function mutations are strongly correlated with the loss of ARID1A protein expression in endometriosis-associated ovarian cancer 6 , 7 . On the other hand, our recent genomic study clarified that  ARID1A  loss-of-function mutations are detected in 13% of ovarian endometriosis cases 14 . Some deep infiltrating endometriosis cases also harbor  ARID1A  mutations 15 , 16 . Although several previous studies demonstrated that ARID1A was expressed in endometriosis by immunohistochemical analysis 17 – 22 , the mutation status of  ARID1A  in endometriotic epithelial cells was not investigated. The association of ARID1A protein expression with  ARID1A  mutations in benign endometriosis remains unclear.\nIn this study, we performed immunohistochemical staining for ARID1A in ovarian endometriosis samples whose  ARID1A  mutation status was determined by whole-exome sequencing or target gene sequencing to clarify the correlation between ARID1A protein expression and the  ARID1A  mutation status in ovarian endometriosis. Additionally, we evaluated an association between ARID1A protein expression and the  ARID1A  mutation status in ovarian clear cell carcinomas by immunohistochemical analysis. We demonstrated that ARID1A protein expression was retained in all ovarian endometriosis samples and a small portion of ovarian clear cell carcinoma samples harboring  ARID1A  loss-of-function mutations.\n\nWe assessed ARID1A immunoreactivity in 15 frozen section samples derived from six ovarian endometriosis patients (Fig.  1 ). We performed multiregional sampling from ovarian endometriosis tissues in three patients (Table  1 ). A representative image of ARID1A immunostaining for a multisampling case (ENDO_3) is shown in Fig.  2 . Both the  ARID1A  wild-type region and the  ARID1A  mutated regions showed positive immunoreactivity for ARID1A. Immunohistochemical analysis demonstrated positive immunoreactivity for ARID1A in all eight frozen section samples harboring  ARID1A  loss-of-function mutations and seven frozen section samples without  ARID1A  mutations. Table  1  shows the mutation status of other cancer-associated genes in samples with  ARID1A  mutations. While mutations in oncogenes such as  PIK3CA  and  KRAS  were detected in two samples (ENDO1 and ENDO3), mutations in tumor suppressor genes such as  PTEN ,  ATM , and  TP53  were not detected in any of the 15 ovarian endometriosis samples. Figure 1 Final analysis set of ovarian endometriosis in this study. We recruited 54 ovarian endometriosis patients for which whole-exome sequencing or target gene sequencing was conducted in our previous study 14 . Then, we collected 16 frozen tissue section samples from 7 patients with  ARID1A  mutations and 69 formalin-fixed paraffin-embedded (FFPE) samples from 47 patients without  ARID1A  mutations. Table 1 List of ovarian endometriosis samples showing the  ARID1A  mutation status and ARID1A protein expression. Patient Sampling site ARID1A  mutation MAF PIK3CA  mutation MAF KRAS  mutation MAF ARID1A protein expression ENDO_1 E1 p.G1711fs 0.46 p.C378F 0.37 p.Q61H 0.45 Positive ENDO_2 E1 WT Positive E2 Q537X 0.25 Positive E3 WT Positive E4 WT Positive ENDO_3 E1 E1733X 0.51 p.G118D 0.36 p.G12D 0.44 Positive E2 E1733X 0.31 p.G118D 0.48 p.G12D 0.33 Positive E3 E1733X 0.31 p.G118D 0.39 p.G12D 0.30 Positive E4 WT Positive ENDO_4 E1 S825fs 0.37 Positive ENDO_5 E1 Q1493X 0.34 Positive E2 WT Positive E3 WT Positive E4 WT Positive ENDO_6 E1 p.F2208fs 0.42 Positive Figure 2 ARID1A protein expression in ovarian endometriosis (ENDO_3).  (A)  Multisampling sites in a unilateral ovarian endometriosis lesion are shown.  (B)  ARID1A protein expression was observed in not only one  ARID1A  wild-type sample but also three  ARID1A  p.E1733X samples. The scale bars represent 100 µm.\nFinal analysis set of ovarian endometriosis in this study. We recruited 54 ovarian endometriosis patients for which whole-exome sequencing or target gene sequencing was conducted in our previous study 14 . Then, we collected 16 frozen tissue section samples from 7 patients with  ARID1A  mutations and 69 formalin-fixed paraffin-embedded (FFPE) samples from 47 patients without  ARID1A  mutations.\nList of ovarian endometriosis samples showing the  ARID1A  mutation status and ARID1A protein expression.\nARID1A protein expression in ovarian endometriosis (ENDO_3).  (A)  Multisampling sites in a unilateral ovarian endometriosis lesion are shown.  (B)  ARID1A protein expression was observed in not only one  ARID1A  wild-type sample but also three  ARID1A  p.E1733X samples. The scale bars represent 100 µm.\nNext, we assessed ARID1A protein expression in 63 FFPE samples derived from 41 ovarian endometriosis patients without  ARID1A  mutations (Fig.  1 ). As expected, ARID1A protein expression was detected in all 63 FFPE tissue samples. In summary, all 78 endometriosis samples retained ARID1A protein expression regardless of the  ARID1A  mutation status.\nTo clarify the significance of  ARID1A  loss-of-function mutations in endometriosis, we compared the clinicopathological features of ovarian endometriosis patients with  ARID1A  loss-of-function mutations to those without  ARID1A  mutations (Supplementary Table  1 ). Interestingly, ovarian endometriosis patients with  ARID1A  loss-of-function mutations had a higher frequency of endometriosis lesions in bilateral ovaries ( P  = 0.006). There were no differences in other characteristics according to the  ARID1A  mutation status (Supplementary Table  1 ).\nTo evaluate an association between ARID1A protein expression and the  ARID1A  mutation status, we performed immunohistochemical analysis for 99 ovarian clear cell carcinoma samples whose  ARID1A  mutation status was already investigated in our previous study (Supplementary Table  2 )  12 . Nine clear cell carcinomas with  ARID1A  mutations and four clear cell carcinomas without  ARID1A  mutations were excluded from this analysis because of the low antigenicity or low quality of FFPE section samples (Fig.  3 ). Of 60 clear cell carcinoma samples with  ARID1A  mutations, 49 (81.7%) showed a loss of ARID1A protein expression (Table  2 ). Specifically, 47 of 56 samples (83.9%) with  ARID1A  loss-of-function mutations showed a loss of ARID1A protein expression. On the other hand, 6 of 26 samples (23.1%) without  ARID1A  mutations also demonstrated a loss of ARID1A protein expression. The presence of  ARID1A  loss-of-function mutations was significantly associated with the loss of ARID1A protein expression in clear cell carcinomas ( P  < 0.001) (Fig.  4 A) and the representative ARID1A staining images correspond to four patterns on the basis of  ARID1A  mutations and ARID1A protein expression (Fig.  4 B).\n Figure 3 Final analysis set of ovarian clear cell carcinoma in this study. We enrolled 99 patients with ovarian clear cell carcinoma which were already sequenced in our previous study 12 . Additionally, we prepared FFPE tissue sections from 99 clear cell carcinoma cases for immunohistochemical analysis. Table 2 Association of ARID1A protein expression with the  ARID1A  mutation pattern in ovarian clear cell carcinoma. ARID1A  mutation pattern Number ARID1A protein expression Loss Positive Two or more nonsense and/or indel mutations 15 14 1 One nonsense or one indel mutation 40 32 8 One indel mutation and one silent mutation 1 1 0 One missense mutation 1 0 1 One splicing mutation 3 2 1 Total 60 49 11 Figure 4 The association between  ARID1A  loss-of-function mutations and ARID1A protein expression in ovarian clear cell carcinomas.  (A)  The number of clear cell carcinomas with or without ARID1A protein expression and/or  ARID1A  mutations is shown.  (B)  Representative ARID1A staining images correspond to four patterns on the basis of  ARID1A  mutations and ARID1A protein expression. The scale bars represent 100 µm.\nFinal analysis set of ovarian clear cell carcinoma in this study. We enrolled 99 patients with ovarian clear cell carcinoma which were already sequenced in our previous study 12 . Additionally, we prepared FFPE tissue sections from 99 clear cell carcinoma cases for immunohistochemical analysis.\nAssociation of ARID1A protein expression with the  ARID1A  mutation pattern in ovarian clear cell carcinoma.\nThe association between  ARID1A  loss-of-function mutations and ARID1A protein expression in ovarian clear cell carcinomas.  (A)  The number of clear cell carcinomas with or without ARID1A protein expression and/or  ARID1A  mutations is shown.  (B)  Representative ARID1A staining images correspond to four patterns on the basis of  ARID1A  mutations and ARID1A protein expression. The scale bars represent 100 µm.\nWe examined the correlation of  ARID1A  loss-of-function mutations with the loss of ARID1A protein expression. Figure  5  depicts the correlation of  ARID1A  allelic imbalance or the number of  ARID1A  loss-of-function mutations with ARID1A protein expression. All samples that harbored  ARID1A  allelic imbalance showed a loss of ARID1A protein expression. In addition, 14 of 15 samples that harbored multiple loss-of-function mutations showed a loss of ARID1A protein expression. Figure 5 Strong correlation between  ARID1A  allelic imbalance or the number of  ARID1A  loss-of-function mutations and ARID1A protein expression in clear cell carcinoma. The heatmap shows the landscape of the  ARID1A  mutation status, allelic imbalance, ARID1A protein expression and other cancer-associated gene mutations in each clear cell carcinoma sample.\nStrong correlation between  ARID1A  allelic imbalance or the number of  ARID1A  loss-of-function mutations and ARID1A protein expression in clear cell carcinoma. The heatmap shows the landscape of the  ARID1A  mutation status, allelic imbalance, ARID1A protein expression and other cancer-associated gene mutations in each clear cell carcinoma sample.\nARID1A protein expression was observed in 9 of 56 clear cell carcinoma samples (16.1%) with  ARID1A  loss-of-function mutations. Because we could not use serial sections for both immunohistochemical analysis in this study and target gene sequencing in the previous study, we validated the  ARID1A  mutation status of FFPE tissue samples in nine clear cell carcinoma samples with  ARID1A  truncating mutations. We macrodissected cancer cells, extracted DNA, and performed Sanger sequencing for  ARID1A  (Table  3 ). Although PCR was not successful in one sample due to poor DNA quality, we validated that seven FFPE samples harbored  ARID1A  loss-of-function mutations. In only one sample, the targeted  ARID1A  mutation was not detected by Sanger sequencing, probably because the mutation was in a subclonal state (MAF = 0.27). Table 3 Validation of the  ARID1A  mutation status using FFPE samples from clear cell carcinomas with  ARID1A  loss-of-function mutations. Patient ARID1A  mutation MAF ARID1A protein expression Validation OCCC_8 p.Y2148X 0.45 Positive p.Y2148X OCCC_23 p.Q393X 0.27 Positive Normal OCCC_50 p.R1461X 0.55 Positive PCR failure OCCC_61 p.Y395X 0.4 Positive p.Y395X OCCC_67 p.R1772X 0.31 Positive p.R1772X OCCC_74 p.Y1431X 0.46 Positive p.Y1431X OCCC_76 p.Q1454X/p.F1823fs 0.22/0.23 Positive p.Q1454X/p.F1823fs OCCC_77 p.Q393X 0.24 Positive p.Q393X OCCC_98 p.R727fs 0.31 Positive p.R727fs\nValidation of the  ARID1A  mutation status using FFPE samples from clear cell carcinomas with  ARID1A  loss-of-function mutations.\nTo confirm concordance of the ARID1A staining level between frozen sections and FFPE samples, we prepared frozen sections and FFPE samples from the same patient. ARID1A immunohistochemical staining of frozen sections was similar to that of FFPE samples in two clear cell carcinomas and ovarian endometriosis case (Supplementary Fig.  2 ).\nFinally, we compared the clinicopathological features of ovarian clear cell carcinoma with  ARID1A  loss-of-function mutations to those of ovarian clear cell carcinoma without  ARID1A  mutations (Supplementary Table  3 ). Although the optimal rate of primary debulking surgery was marginally lower in the  ARID1A  loss-of-function mutation group than in the  ARID1A  wild-type group ( P  = 0.052), no significant differences in any clinicopathological characteristics, including prognosis, were observed (Supplementary Fig.  3 ).\n\nStrong evidence for an association between ovarian endometriosis and ovarian clear cell and endometrioid carcinomas has been established in many studies 23 – 26 . In particular, there is epidemiological evidence that a personal history of endometriosis increases the risk of clear cell and endometrioid carcinomas 27 – 29 . Pathological studies have also demonstrated that atypical endometriosis merging between endometriosis and carcinoma exists in ovarian clear cell and endometrioid carcinoma cases 30 , 31 . In addition, there is accumulating molecular evidence linking endometriosis with clear cell carcinoma 7 , 14 , 26 . Wiegand et al. demonstrated that the loss of ARID1A caused by  ARID1A  loss-of-function mutations is observed in clear cell carcinoma and contiguous atypical endometriosis but not in distant endometriosis 7 . It is well known that  ARID1A  mutations are frequently detected in ovarian clear cell and endometrioid carcinomas but not in high-grade serous ovarian carcinomas 7 , 12 , 28 , 32 . These results suggest that  ARID1A  loss-of-function mutations are a driver event in endometriosis-associated ovarian cancer 6 , 7 . On the other hand, our recent studies clarified that cancer-associated genes such as  ARID1A ,  PIK3CA  and  KRAS  are frequently mutated not only in ovarian clear cell carcinoma but also in ovarian endometriosis 12 , 14 . Specifically,  ARID1A  loss-of-function mutations were detected in 7 of 54 ovarian endometriosis patients in our previous study 14 . Additionally, ovarian endometriosis samples harboring a single  ARID1A  loss-of-function mutation had mutations in oncogenes such as  PIK3CA  and  KRAS  and maintained benign conditions pathologically (Table  1 ) 14 . On the other hand, mutations in tumor suppressor genes, such as  PTEN ,  ATM , and  TP53 , were not detected in these ovarian endometriosis samples. The significance of  ARID1A  mutations in the malignant transformation of ovarian endometriosis remains unclear.\nSeveral studies have focused on ARID1A protein expression in endometriosis or ovarian cancer. Immunohistochemical analyses of ARID1A in ovarian cancer demonstrated that 0–40% of endometriosis lesions adjacent to ovarian cancer showed a loss of ARID1A protein expression, whereas all distant endometriosis lesions in ovarian cancer expressed ARID1A 7 , 17 , 19 , 21 , 33 . Similarly, several studies showed that ARID1A was expressed in almost all benign endometriosis lesions if ARID1A protein expression in stromal cells was correctly assessed as an internal positive control 17 – 22 . Although immunohistochemical staining and assessment protocols were not unified between studies, there was an obvious difference in ARID1A protein expression between benign endometriosis and endometriosis-associated ovarian cancer. These findings suggest that the loss of ARID1A protein expression might be an early driver event in the malignant transformation of ovarian endometriosis. However, the mutation status of  ARID1A  in endometriotic epithelial cells was not examined in these studies. Furthermore, the mechanism by which ARID1A protein expression is lost has not been sufficiently discussed. Wiegand et al. showed that 25% of clear cell carcinomas with loss-of-function mutations in one  ARID1A  allele retained ARID1A protein expression 7 . They also found that both mutant and wild-type alleles of  ARID1A  were expressed by using RNA sequencing data derived from nine clear cell carcinomas with  ARID1A  loss-of-function mutations. Based on these results, Wiegand et al. concluded that  ARID1A  could function as a haploinsufficient tumor suppressor. On the other hand, our study demonstrated that ARID1A protein expression was retained in 16% of clear cell carcinomas harboring  ARID1A  loss-of-function mutations (Fig.  4 A). Moreover, ARID1A protein expression was retained in all benign endometriosis samples with  ARID1A  loss-of-function mutations. These findings are inconsistent with the concept of haploinsufficiency proposed by Wiegand et al. 7 . Our study also demonstrated that 14 of 15 samples that harbored multiple loss-of-function mutations showed a loss of ARID1A protein expression. In particular, all clear cell carcinoma samples harboring both  ARID1A  loss-of-function mutations and  ARID1A  allelic imbalance showed a loss of ARID1A protein expression. These findings suggest that the “two-hit” hypothesis can explain the cause of ARID1A loss in cancer cells 34 – 36 . In Knudson’s two-hit hypothesis 34 , germline mutation in tumor suppressor gene lead to a hereditary susceptibility to cancer and the inactivation of both alleles of tumor suppressor genes is essential to cause a phenotypic chance, leading to carcinogenesis. In other words, the “two-hit” hypothesis can explain why ARID1A protein expression was retained in all benign endometriosis samples with  ARID1A  loss-of-function mutations and a portion of clear cell carcinoma with  ARID1A  loss-of-function mutations. Taken together, these results suggest that the two-hit would be necessary for benign endometriosis with  ARID1A  heterozygous mutation to transform into malignant tumor.\nConsistent with Wiegand et al. 7 , we also observed a portion of clear cell carcinomas without  ARID1A  mutations showed a loss of ARID1A protein expression, suggesting that epigenetic silencing, posttranscriptional and posttranslational regulation as well as genomic alterations might be important for the loss of ARID1A protein expression in clear cell carcinoma.\nIn this study, the sample size of ovarian endometriosis patients with  ARID1A  mutations was limited. It is necessary to assess ARID1A protein expression in ovarian endometriosis samples with  ARID1A  mutations in independent data sets. Although we used serial sections to assess  ARID1A  mutations and ARID1A protein expression in endometriosis, we could not extract DNA, RNA and protein from the same tissue simultaneously. There may be room for improvement not only in the number of samples but also in the extraction of DNA/RNA/protein for further study.\nIn conclusion, we clarified that ARID1A protein expression was retained in ovarian endometriosis samples harboring  ARID1A  loss-of-function mutations. The mechanism of ARID1A loss, which occurs specifically in endometriosis-associated ovarian cancer but not in ovarian endometriosis, is an important key for elucidating the pathogenesis of the malignant transformation of ovarian endometriosis.\n\nThis study was performed in conformity with the Declaration of Helsinki and approved by the institutional ethics review boards of Niigata University, Niigata Chuo General Hospital, and the National Institute of Genetics. All patients provided written informed consent for the collection of samples and subsequent analyses.\nWe recruited 54 ovarian endometriosis patients for which whole-exome sequencing or target gene sequencing was conducted in our previous study 14 . We defined frameshift indels mutations, and nonsense mutations as  ARID1A  loss-of-function mutations. Then, we collected 16 frozen tissue section samples from seven patients with  ARID1A  mutations and 69 formalin-fixed paraffin-embedded (FFPE) samples from 47 patients without  ARID1A  mutations (Fig.  1 ). Frozen tissue samples were obtained from the same tissue blocks used for sequencing in our previous study 14 . Of these samples, one frozen tissue sample and six FFPE tissue samples were excluded from this study because there were no endometriotic epithelial cells in either the frozen tissue or FFPE sample.\nWe also enrolled 99 patients with ovarian clear cell carcinoma in this study to compare the association between ARID1A protein expression and the  ARID1A  mutation status with that in ovarian endometriosis. These clear cell carcinoma samples were already sequenced in our previous study 12 . Sixty-nine of 99 (69.7%) ovarian clear cell carcinoma samples harbored  ARID1A  mutations (Supplementary Table  2 ). Additionally, we prepared FFPE tissue sections from 99 clear cell carcinoma cases for immunohistochemical analysis (Fig.  3 ). We also prepared frozen tissue sections from two clear cell carcinomas to assess the concordance of ARID1A immunoreactivity between frozen tissue and FFPE samples in the same patient.\nHematoxylin and eosin-stained sections of all tissues used in this study were histologically reviewed by an experienced gynecologic pathologist (T.M.). All frozen tissue samples were cut from surgical specimens, embedded in Tissue-Tek O.C.T. compound (Sakura Finetek, Torrance, CA, USA) in a Tissue-Tek Cryomold (Sakura Finetek) and quickly frozen in liquid nitrogen as described in our previous study 14 .\nImmunohistochemical analysis of ARID1A protein expression was performed for frozen tissue section and FFPE tissue section samples. A polyclonal rabbit anti-ARID1A antibody (HPA005456, Sigma-Aldrich, St. Louis, MO, USA) was used for immunostaining as a primary antibody. Frozen tissue sections (6 µm) and FFPE tissue sections (5 µm) were cut with a cryostat and a microtome, respectively. FFPE tissue sections were stained as previously described 37 , 38 . Briefly, after deparaffinization, antigen retrieval was carried out with Target Retrieval Solution (10 mM citrate buffer, pH 6.0; Dako, Tokyo, Japan) in a microwave for 20 min at 98 °C. Subsequently, the sections were incubated with the primary antibody (1:500 dilution) overnight and biotinylated secondary antibodies (Vector Laboratories, Burlingame, CA, USA) for 1 h, followed by incubation with ABC reagent (Dako) and 3,3′-diaminobenzidine (Sigma-Aldrich) for 3 min. Slides were counterstained with hematoxylin.\nWe fixed frozen tissue sections with 4% paraformaldehyde at 4 °C for 20 min followed by methanol at − 20 °C for 10 min. The immunohistochemical staining protocol after fixation was the same as the protocol for FFPE tissue sections.\nWe assessed normal nonepithelial cells, including endothelial cells, fibroblasts, and lymphocytes, as positive internal controls. The immunostaining was decided as positive if epithelial cells showed definite nuclear staining by two investigators (Y.N. and R.T.) The distribution of the percentage of positive cells showed bimodality as a previous study 39 . We evaluated samples with more than 80% positive cells as ARID1A positive and samples with under 20% positive cells as ARID1A loss (Supplementary Fig.  1 ). Samples in which normal cells in the stroma had no immunoreactivity were defined as having low antigenicity or low quality and excluded from the subsequent analysis.\nTo validate the mutation status of ovarian clear cell carcinoma FFPE samples, we prepared FFPE serial section following the one used for immunohistochemistry assay to perform Sanger sequencing per FFPE sample. We isolated tumor cells by needle macrodissection and extracted DNA using a QIAamp DNA FFPE Tissue Kit (QIAGEN Ltd., Manchester, UK) according to the manufacturer’s instructions.\nWe performed polymerase chain reaction (PCR) using a KAPA Taq EXtra HotStart ReadyMix PCR Kit, and the primers used are listed in Supplementary Table  4 . We designed PCR primers using Primer3 software ( https://bioinfo.ut.ee/primer3-0.4.0/ ). PCR products were purified and sequenced by GENEWIZ (Saitama, Japan).\nWe conducted all standard statistical tests with the R program ( https://www.r-project.org ). We compared categorical variables between two groups by Fisher’s exact test and continuous variables between two groups by the Wilcoxon rank-sum test. Progression-free survival (PFS) and overall survival (OS) were estimated using the Kaplan–Meier method. Deviation in the mutant allele frequency (MAF) of the somatic mutation from 0.5 was assessed by a one-sided binomial test. A  P  value < 0.05 was considered allelic imbalance 40 .\n\nSupplementary Information.\nSupplementary Information.","source_license":"CC0","license_restricted":false}