Super enhancer loci of EGFR regulate EGFR variant 8 through enhancer RNA and strongly associate with survival in HNSCCs.

Epidermal growth factor receptor (EGFR) has been shown to be overexpressed in human cancers due to mutation, amplification, and epigenetic hyperactivity, which leads to deregulated transcriptional mechanism. Among the eight different EGFR isoforms, the mechanism of regulation of full-length variant 1 is well-known, no studies have examined the function & factors regulating the expression of variant 8. This study aimed to understand the function of EGFR super-enhancer loci and its associated transcription factors regulating the expression of EGFR variant 8. Our study shows that overexpression of variant 8 and its transcription was more prevalent than variant 1 in many cancers and positively correlated with the EGFR-AS1 expression in oral cancer and HNSCC. Notably, individuals overexpressing variant 8 showed shorter overall survival and had a greater connection with other clinical traits than patients with overexpression of variant 1. In this study, TCGA enhancer RNA profiling on the constituent enhancer (CE1 and CE2) region revealed that the multiple enhancer RNAs formed from CE2 by employing CE1 as a promoter. Our bioinformatic analysis further supports the enrichment of enhancer RNA specific chromatin marks H3K27ac, H3K4me1, POL2 and H2AZ on CE2. GeneHancer and 3D chromatin capture analysis showed clustered interactions between CE1, CE2 loci and this interaction may regulates expression of both EGFR-eRNA and variant 8. Moreover, increased expression of SNAI2 and its close relationship to EGFR-AS1 and variant 8 suggest that SNAI2 could regulates variant 8 overexpression by building a MegaTrans complex with both EGFR-eRNA and EGFR-AS1. Our findings show that EGFR variant 8 and its transcriptional regulation & chromatin modification by eRNAs may provide a rationale for targeting RNA splicing in combination with targeted EGFR therapies in cancer.

Noninvasive graft monitoring using donor-derived cell-free DNA in Japanese liver transplantation.

AIM: To evaluate the use of donor-derived cell-free DNA (dd-cfDNA) in diagnosing graft injuries in Japanese liver transplantation (LTx), including family-related living donors. METHODS: A total of 321 samples from 10 newly operated LTx recipients were collected to monitor the early dynamics of dd-cfDNA levels after LTx. Fifty-five samples from 55 recipients were collected during protocol biopsies (PB), whereas 36 samples from 27 recipients were collected during event biopsies, consisting of 11 biopsy-proven acute rejection (AR), 20 acute dysfunctions without rejection (ADWR), and 5 chronic rejections. The levels of dd-cfDNA were quantified using a next-generation sequencer based on single nucleotide polymorphisms. RESULTS: The dd-cfDNA levels were elevated significantly after LTx, followed by a rapid decline to the baseline in patients without graft injury within 30 days post-LTx. The dd-cfDNA levels were significantly higher in the 11 samples obtained during AR than those obtained during PB (p < 0.0001), which decreased promptly after treatment. The receiver operator characteristic curve analysis of diagnostic ability yielded areas under the curve of 0.975 and 0.897 for AR (rejection activity index [RAI] ≥3) versus PB and versus non-AR (ADWR + PB). The dd-cfDNA levels during AR were elevated earlier and correlated more strongly with the RAI (r = 0.740) than aspartate aminotransferase/alanine aminotransferase. The dd-cfDNA levels were neither associated with graft fibrosis based on histology nor the status of donor-specific antibodies in PB samples. CONCLUSIONS: Donor-derived cell-free DNA serves as a sensitive biomarker for detecting graft injuries in LTx. Further large-scale cohort studies are warranted to optimize its use in differentiating various post-LTx etiologies.

Detection of APP gene recombinant in human blood plasma.

The pathogenesis of Alzheimer's disease (AD) is believed to involve the accumulation of amyloid-ß in the brain, which is produced by the sequential cleavage of amyloid precursor protein (APP) by ß-secretase and γ-secretase. Recently, analysis of genomic DNA and mRNA from postmortem brain neurons has revealed intra-exonic recombinants of APP (gencDNA), which have been implicated in the accumulation of amyloid-ß. In this study, we computationally analyzed publicly available sequence data (SRA) using probe sequences we constructed to screen APP gencDNAs. APP gencDNAs were detected in SRAs constructed from both genomic DNA and RNA obtained from the postmortem brain and in the SRA constructed from plasma cell-free mRNA (cf-mRNA). The SRA constructed from plasma cf-mRNA showed a significant difference in the number of APP gencDNA reads between SAD and NCI: the p-value from the Mann-Whitney U test was 5.14 × 10-6. The transcripts were also found in circulating nucleic acids (CNA) from our plasma samples with NGS analysis. These data indicate that transcripts of APP gencDNA can be detected in blood plasma and suggest the possibility of using them as blood biomarkers for Alzheimer's disease.

Spatial genomic diversity associated with APOBEC mutagenesis in squamous cell carcinoma arising from ovarian teratoma.

Although the gross and microscopic features of squamous cell carcinoma arising from ovarian mature cystic teratoma (MCT-SCC) vary from case to case, the spatial spreading of genomic alterations within the tumor remains unclear. To clarify the spatial genomic diversity in MCT-SCCs, we performed whole-exome sequencing by collecting 16 samples from histologically different parts of two MCT-SCCs. Both cases showed histological diversity within the tumors (case 1: nonkeratinizing and keratinizing SCC and case 2: nonkeratinizing SCC and anaplastic carcinoma) and had different somatic mutation profiles by histological findings. Mutation signature analysis revealed a significantly enriched apolipoprotein B mRNA editing enzyme catalytic subunit (APOBEC) signature at all sites. Intriguingly, the spread of genomic alterations within the tumor and the clonal evolution patterns from nonmalignant epithelium to cancer sites differed between cases. TP53 mutation and copy number alterations were widespread at all sites, including the nonmalignant epithelium, in case 1. Keratinizing and nonkeratinizing SCCs were differentiated by the occurrence of unique somatic mutations from a common ancestral clone. In contrast, the nonmalignant epithelium showed almost no somatic mutations in case 2. TP53 mutation and the copy number alteration similarities were observed only in nonkeratinizing SCC samples. Nonkeratinizing SCC and anaplastic carcinoma shared almost no somatic mutations, suggesting that each locally and independently arose in the MCT. We demonstrated that two MCT-SCCs with different histologic findings were highly heterogeneous tumors with clearly different clones associated with APOBEC-mediated mutagenesis, suggesting the importance of evaluating intratumor histological and genetic heterogeneity among multiple sites of MCT-SCC.

AMBRA1 p.Gln30Arg Mutation, Identified in a Cowden Syndrome Family, Exhibits Hyperproliferative Potential in hTERT-RPE1 Cells.

Cowden syndrome (CS) is a rare autosomal dominant disorder associated with multiple hamartomatous and neoplastic lesions in various organs. Most CS patients have been found to have germline mutations in the PTEN tumor suppressor. In the present study, we investigated the causative gene of CS in a family of PTEN (phosphatase and tensin homolog deleted on chromosome 10) -negative CS patients. Whole exome sequencing analysis revealed AMBRA1 (Autophagy and Beclin 1 Regulator 1) as a novel candidate gene harboring two germline variants: p.Gln30Arg (Q30R) and p.Arg1195Ser (R1195S). AMBRA1 is a key regulator of the autophagy signaling network and a tumor suppressor. To functionally validate the role of AMBRA1 in the clinical manifestations of CS, we generated AMBRA1 depletion and Q30R mutation in hTERT-RPE1 (humanTelomerase Reverse Transcriptase-immortalized Retinal Pigmented Epithelial cells) using the CRISPR-Cas9 gene editing system. We observed that both AMBRA1-depleted and mutant cells showed accumulation in the S phase, leading to hyperproliferation, which is a characteristic of hamartomatous lesions. Specifically, the AMBRA1 Q30R mutation disturbed the G1/S transition of cells, leading to continuous mitotic entry of mutant cells, irrespective of the extracellular condition. From our analysis of primary ciliogenesis in these cells, we speculated that the mitotic entry of AMBRA1 Q30R mutants could be due to non-functional primary cilia that lead to impaired processing of extracellular sensory signals. Additionally, we observed a situs inversus phenotype in ambra1-depleted zebrafish, a developmental abnormality resulting from dysregulated primary ciliogenesis. Taken together, we established that the AMBRA1 Q30R mutation that we observed in CS patients might play an important role in inducing the hyperproliferative potential of cells through regulating primary ciliogenesis.

The landscape of genetic alterations of UVB-induced skin tumors in DNA repair-deficient mice.

Non-melanoma skin cancer (NMSC) is mainly caused by ultraviolet (UV)-induced somatic mutations and is characterized by UV signature modifications. Xeroderma pigmentosum group A (Xpa) knockout mice exhibit extreme UV-induced photo-skin carcinogenesis, along with a photosensitive phenotype. We performed whole-exome sequencing (WES) of squamous cell carcinoma (SCC) samples after repetitive ultraviolet B (UVB) exposure to investigate the differences in the landscape of somatic mutations between Xpa knockout and wild-type mice. Although the tumors that developed in mice harboured UV signature mutations in a similar set of cancer-related genes, the pattern of transcriptional strand asymmetry was largely different; UV signature mutations in Xpa knockout and wild-type mice preferentially occurred in transcribed and non-transcribed strands, respectively, reflecting a deficiency in transcription-coupled nucleotide excision repair in Xpa knockout mice. Serial time point analyses of WES for a tumor induced by only a single UVB exposure showed pathogenic mutations in Kras, Fat1, and Kmt2c, which may be driver genes for the initiation and promotion of SCC in Xpa knockout mice. Furthermore, the inhibitory effects on tumor production in Xpa knockout mice by the anti-inflammatory CXCL1 monoclonal antibody affected the pattern of somatic mutations, wherein the transcriptional strand asymmetry was attenuated and the activated signal transduction was shifted from the RAS/RAF/MAPK to the PIK3CA pathway.

High incidence of PI3K pathway gene mutations in South Indian cervical cancers.

INTRODUCTION: Cervical cancer is the second most common cancer in India. The phosphatidylinositol-3 kinase (PI3K) signaling is one of the most commonly activated pathways in cancer and comprises key molecules commonly targeted in cancer therapy. This study analyzed six PI3K pathway gene mutations. METHODS: We carried out targeted next-generation sequencing of six PI3K pathway genes (PIK3CA, PIK3R1, PTEN, AKT1, TSC2, and mTOR) in a total of 93 South Indian cervical cancer samples and confirmed them by sanger sequencing. RESULTS: The PI3K pathway gene mutations were observed in 54.8% (51/93) of the tumors and PIK3CA was the most mutated (34.4%, 32/93), followed by TSC2 (18.3%, 17/93), and PIK3R1 (14%, 13/93). The PIK3CA hotspot mutations E542K and E545K observed in this study were likely to disrupt the p110α-p85α interaction that could result in the PI3K pathway activation. We also found a few novel mutations in PIK3R1, PTEN, AKT1, TSC2, and mTOR genes while some of the tumors harbored multiple mutations in the genes of the PI3K pathway. The majority of the tumors were positive for high-risk HPV16/18 (60.7%). CONCLUSIONS: The high incidence of the PI3K pathway gene mutations observed in this study could be exploited for the therapeutic management of cervical cancers.

Movements of Ancient Human Endogenous Retroviruses Detected in SOX2-Expressing Cells.

Human endogenous retroviruses (HERVs) occupy approximately 8% of the human genome. HERVs, transcribed in early embryos, are epigenetically silenced in somatic cells, except under pathological conditions. HERV-K is thought to protect embryos from exogenous viral infection. However, uncontrolled HERV-K expression in somatic cells has been implicated in several diseases. Here, we show that SOX2, which plays a key role in maintaining the pluripotency of stem cells, is critical for HERV-K LTR5Hs. HERV-K undergoes retrotransposition within producer cells in the absence of Env expression. Furthermore, we identified new HERV-K integration sites in long-term culture of induced pluripotent stem cells that express SOX2. These results suggest that the strict dependence of HERV-K on SOX2 has allowed HERV-K to protect early embryos during evolution while limiting the potentially harmful effects of HERV-K retrotransposition on host genome integrity in these early embryos. IMPORTANCE Human endogenous retroviruses (HERVs) account for approximately 8% of the human genome; however, the physiological role of HERV-K remains unknown. This study found that HERV-K LTR5Hs and LTR5B were transactivated by SOX2, which is essential for maintaining and reestablishing pluripotency. HERV-K can undergo retrotransposition within producer cells without env expression, and new integration sites may affect cell proliferation. In induced pluripotent stem cells (iPSCs), genomic impairment due to HERV-K retrotransposition has been identified, but it is a rare event. Considering the retention of SOX2-responsive elements in the HERV-K long terminal repeat (LTR) for over 20 million years, we conclude that HERV-K may play important physiological roles in SOX2-expressing cells.

Spatiotemporal dynamics of clonal selection and diversification in normal endometrial epithelium.

It has become evident that somatic mutations in cancer-associated genes accumulate in the normal endometrium, but spatiotemporal understanding of the evolution and expansion of mutant clones is limited. To elucidate the timing and mechanism of the clonal expansion of somatic mutations in cancer-associated genes in the normal endometrium, we sequence 1311 endometrial glands from 37 women. By collecting endometrial glands from different parts of the endometrium, we show that multiple glands with the same somatic mutations occupy substantial areas of the endometrium. We demonstrate that "rhizome structures", in which the basal glands run horizontally along the muscular layer and multiple vertical glands rise from the basal gland, originate from the same ancestral clone. Moreover, mutant clones detected in the vertical glands diversify by acquiring additional mutations. These results suggest that clonal expansions through the rhizome structures are involved in the mechanism by which mutant clones extend their territories. Furthermore, we show clonal expansions and copy neutral loss-of-heterozygosity events occur early in life, suggesting such events can be tolerated many years in the normal endometrium. Our results of the evolutionary dynamics of mutant clones in the human endometrium will lead to a better understanding of the mechanisms of endometrial regeneration during the menstrual cycle and the development of therapies for the prevention and treatment of endometrium-related diseases.

APOBEC mediated mutagenesis drives genomic heterogeneity in endometriosis.

Endometriosis is a benign gynecologic condition, acting as a precursor of certain histological subtypes of ovarian cancers. The epithelial cells of endometriotic tissues and normal uterine endometrium accumulated somatic mutations in cancer-associated genes such as phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) and Kirsten rat sarcoma (KRAS) proto-oncogene. To determine the genomic characteristic of endometriotic epithelial cells and normal uterine endometrium and to identify the predominant mutational process acting on them, we studied the somatic mutation profiles obtained from whole exome sequencing of 14 endometriotic epithelium and 11 normal uterine endometrium tissues and classified them into mutational signatures. We observed that single base substitutions 2/13 (SBS), attributed to Apolipoprotein B mRNA Editing Enzyme Catalytic Subunit (APOBEC) induced mutagenesis, were significant in endometriotic tissues, but not in the normal uterine endometrium. Additionally, the larger number and wider allele frequency distribution of APOBEC signature mutations, compared to cancer-associated driver mutations in endometriotic epithelium suggested APOBEC mutagenesis as an important source of mutational burden and heterogeneity in endometriosis. Further, the relative risk of enriched APOBEC signature mutations was higher in endometriosis patients who were carriers of APOBEC3A/3B germline deletion, a common polymorphism in East Asians which involves the complete loss of APOBEC3B coding region. Our results illustrate the significance of APOBEC induced mutagenesis in driving the genomic heterogeneity of endometriosis.

Proposing a molecular classification associated with hypercoagulation in ovarian clear cell carcinoma.

BACKGROUND: Although ovarian clear cell carcinoma (CCC) is associated with high incidence of thromboembolism, the clinicopathological and biological significance of hypercoagulable status in CCC remains unclear. MATERIALS AND METHODS: We retrospectively analyzed pretreatment D-dimer levels, thromboembolic status, and clinical outcome of 125 CCCs in the discovery set and 143 CCCs in two other independent validation sets. Next, we performed RNA sequencing of 93 CCCs and compared coagulation-related gene profiles with 2492 pan-cancer data. We investigated differences in molecular characteristics of CCC subclasses based on coagulation status. RESULTS: In the discovery dataset, D-dimer elevation above the normal range was significantly associated with shorter progression-free and overall survival, irrespective to thromboembolic status. Multivariate analysis identified D-dimer elevation and clinical stage as an independent prognostic factors. We confirmed the prognostic significance of D-dimer elevation in the validation sets. Tissue factor and IL6, which are considered key elements of cancer-induced hypercoagulation, were highly expressed in CCC than in other cancers regardless of D-dimer level. Higher activity of various oncogenic pathways was observed in CCC with compared to without D-dimer elevation. Moreover, hierarchical cluster analysis divided 57 CCCs with D-dimer elevation into immunologically hot and cold tumor subtypes. Hot tumors were characterized by enrichment of T-cell inflamed phenotype, inflammation, the epithelial-mesenchymal transition, and high serum levels of CRP, and cold tumors by enrichment of cell cycle and MYC pathways. CONCLUSIONS: CCC represents hypercoagulable disease and elevate D-dimer is a prognostic factor for decreased survival in CCC. D-dimer high CCC has distinct molecular characteristics into the inflammatory-driven pathway (hot tumor) and the immune-suppressive pathway (cold tumor). Treatment implication of our proposed molecular classification merits further investigation.

Significance of Mitochondrial DNA Haplogroup on Epidermal Growth Factor Receptor Mutation in Japanese Patients With Lung Adenocarcinoma.

BACKGROUND/AIM: The frequency of somatic mutations of epidermal growth factor receptor (EGFR) in primary lung adenocarcinoma varies among populations and countries. The aim of the present study was to clarify whether the frequency of EGFR mutations in patients with lung adenocarcinoma depends on their mitochondrial DNA haplogroup, which reflects their maternal lineage. PATIENTS AND METHODS: Using normal lung tissue specimens, the mitochondrial DNA haplogroup was determined by multiplex polymerase chain reaction in 135 Japanese patients who underwent surgery for primary lung adenocarcinoma. RESULTS: The 135 patients were divided into two groups according to the two primitive haplotypes (N group, n=32; M group, n=103). The frequency of EGFR mutations in the N group was significantly higher than that in the M group (69% vs. 48%, p=0.044). The difference was prominent when the analysis was restricted to non-smokers (95% vs. 57%, p<0.01). CONCLUSION: The frequency of EGFR mutations in lung adenocarcinoma patients depends on their mitochondrial lineage.

Substantial anti-gout effect conferred by common and rare dysfunctional variants of URAT1/SLC22A12.

OBJECTIVES: Gout, caused by chronic elevation of serum uric acid levels, is the commonest form of inflammatory arthritis. The causative effect of common and rare variants of ATP-binding cassette transporter G2 (ABCG2/BCRP) on gout risk has been studied, but little attention has been paid to the effect of common (rs121907892, p.W258X) and rare variants of urate transporter 1 (URAT1/SLC22A12) on gout, despite dysfunctional variants of URAT1 having been identified as pathophysiological causes of renal hypouricaemia. METHODS: To address this important but overlooked issue, we investigated the effects of these URAT1 variants on gout susceptibility, using targeted exon sequencing on 480 clinically defined gout cases and 480 controls of Japanese males in combination with a series of functional analyses of newly identified URAT1 variants. RESULTS: Our results show that both common and rare dysfunctional variants of URAT1 markedly decrease the risk of gout (OR 0.0338, reciprocal OR 29.6, P = 7.66 × 10-8). Interestingly, we also found that the URAT1-related protective effect on gout eclipsed the ABCG2-related causative effect (OR 2.30-3.32). Our findings reveal only one dysfunctional variant of URAT1 to have a substantial anti-gout effect, even in the presence of causative variants of ABCG2, a 'gout gene'. CONCLUSION: Our findings provide a better understanding of gout/hyperuricaemia and its aetiology that is highly relevant to personalized health care. The substantial anti-gout effect of common and rare variants of URAT1 identified in the present study support the genetic concept of a 'Common Disease, Multiple Common and Rare Variant' model.

Genetic variant rs10251977 (G>A) in EGFR-AS1 modulates the expression of EGFR isoforms A and D.

Tyrosine kinase inhibitor is an effective chemo-therapeutic drug against tumors with deregulated EGFR pathway. Recently, a genetic variant rs10251977 (G>A) in exon 20 of EGFR reported to act as a prognostic marker for HNSCC. Genotyping of this polymorphism in oral cancer patients showed a similar frequency in cases and controls. EGFR-AS1 expressed significantly high level in tumors and EGFR-A isoform expression showed significant positive correlation (r = 0.6464, p < 0.0001) with reference to EGFR-AS1 expression levels, consistent with larger TCGA HNSCC tumor dataset. Our bioinformatic analysis showed enrichment of alternative splicing marks H3K36me3 and presence of intronic polyA sites spanning around exon 15a and 15b of EGFR facilitates skipping of exon 15b, thereby promoting the splicing of EGFR-A isoform. In addition, high level expression of PTBP1 and its binding site in EGFR and EGFR-AS1 enhances the expression of EGFR-A isoform (r = 0.7404, p < 0.0001) suggesting that EGFR-AS1 expression modulates the EGFR-A and D isoforms through alternative splicing. In addition, this polymorphism creates a binding site for miR-891b in EGFR-AS1 and may negatively regulate the EGFR-A. Collectively, our results suggested the presence of genetic variant in EGFR-AS1 modulates the expression of EGFR-D and A isoforms.

APOBEC: A molecular driver in cervical cancer pathogenesis.

Cervical cancer is one of the foremost common cancers in women. Human papillomavirus (HPV) infection remains a major risk factor of cervical cancer. In addition, numerous other genetic and epigenetic factors also are involved in the underlying pathogenesis of cervical cancer. Recently, it has been reported that apolipoprotein B mRNA editing enzyme catalytic polypeptide like (APOBEC), DNA-editing protein plays an important role in the molecular pathogenesis of cancer. Particularly, the APOBEC3 family was shown to induce tumor mutations by aberrant DNA editing mechanism. In general, APOBEC3 enzymes play a pivotal role in the deamination of cytidine to uridine in DNA and RNA to control diverse biological processes such as regulation of protein expression, innate immunity, and embryonic development. Innate antiviral activity of the APOBEC3 family members restrict retroviruses, endogenous retro-element, and DNA viruses including the HPV that is the leading risk factor for cervical cancer. This review briefly describes the pathogenesis of cervical cancer and discusses in detail the recent findings on the role of APOBEC in the molecular pathogenesis of cervical cancer.

AMBRA1 controls antigen-driven activation and proliferation of naive T cells.

AMBRA1 (activating molecule in Beclin1-regulated autophagy) is a member of the BECN1 (BECLIN1) protein complex, and it plays a role in autophagy, cell death, tumorigenesis and proliferation. We recently reported that on T-cell receptor (TCR) stimulation, AMBRA1 controlled both autophagy and the cell cycle with metabolic regulation. Accumulating evidence has shown that autophagy and metabolic control are pivotal for T-cell activation, clonal expansion and effector/memory cell fate decision. However, it is unknown whether AMBRA1 is involved in T-cell function under physiological conditions. We found that T cells in Ambra1-conditional knockout (cKO) mice induced an exacerbated graft versus host response when they were transplanted into allogeneic BALB/c mice. Furthermore, Ambra1-deficient T cells showed increased proliferation and cytotoxic capability toward specific antigens in response to in vivo stimulation using allogeneic spleen cells. This enhanced immune response mainly contributed to naive T-cell hyperactivity. The T-cell hyperactivity observed in this study was similar to those in some metabolic factor-deficient mice, but not those in other pro-autophagic factor-deficient mice. Under the static condition, however, naive T cells were reduced in Ambra1-cKO mice, the same as in pro-autophagic factor-deficient mice. Collectively, these results suggested that AMBRA1 was involved in regulating T cell-mediated immune responses through autophagy-dependent and -independent mechanisms.

Endogenous retroviruses drive KRAB zinc-finger protein family expression for tumor suppression.

Gene expression aberration is a hallmark of cancers, but the mechanisms underlying such aberrations remain unclear. Human endogenous retroviruses (HERVs) are genomic repetitive elements that potentially function as enhancers. Since numerous HERVs are epigenetically activated in tumors, their activation could cause global gene expression aberrations in tumors. Here, we show that HERV activation in tumors leads to the up-regulation of hundreds of transcriptional suppressors, namely, Krüppel-associated box domain-containing zinc-finger family proteins (KZFPs). KZFP genes are preferentially encoded nearby the activated HERVs in tumors and transcriptionally regulated by these adjacent HERVs. Increased HERV and KZFP expression in tumors was associated with better disease conditions. Increased KZFP expression in cancer cells altered the expression of genes related to the cell cycle and cell-matrix adhesion and suppressed cellular growth, migration, and invasion abilities. Our data suggest that HERV activation in tumors drives the synchronized elevation of KZFP expression, presumably leading to tumor suppression.

ARID1A protein expression is retained in ovarian endometriosis with ARID1A loss-of-function mutations: implication for the two-hit hypothesis.

ARID1A loss-of-function mutation accompanied by a loss of ARID1A protein expression is considered one of the most important driver events in endometriosis-associated ovarian cancer. Although our recent genomic study clarified that ARID1A loss-of-function mutations were detected in 13% of ovarian endometriosis, an association between the ARID1A mutation status and ARID1A protein expression in ovarian endometriosis remains unclear. We performed immunohistochemical staining for ARID1A in 78 ovarian endometriosis samples and 99 clear cell carcinoma samples. We revealed that not only 70 endometriosis samples without ARID1A mutations but also eight endometriosis samples with ARID1A loss-of-function mutations retained ARID1A protein expression. On the other hand, most of clear cell carcinomas with ARID1A loss-of-function mutations showed a loss of ARID1A protein expression. In particular, clear cell carcinoma samples which harbor multiple ARID1A loss-of-function mutations or both a single ARID1A loss-of-function mutation and ARID1A allelic imbalance lost ARID1A protein expression. However, ARID1A protein expression was retained in seven clear cell carcinomas with ARID1A loss-of-function mutations. These results suggest that a single ARID1A loss-of-function mutation is insufficient for ARID1A loss in ovarian endometriosis and some clear cell carcinoma. Further driver events may be needed for the malignant transformation of ovarian endometriosis with ARID1A loss-of-function mutations.

Clonal lineage from normal endometrium to ovarian clear cell carcinoma through ovarian endometriosis.

Clear cell carcinoma of the ovary is thought to arise from endometriosis. In addition, retrograde menstruation of shed endometrium is considered the origin of endometriosis. However, little evidence supports cellular continuity from uterine endometrium to clear cell carcinoma through endometriosis at the genomic level. Here, we performed multiregional whole-exome sequencing to clarify clonal relationships among uterine endometrium, ovarian endometriosis and ovarian clear cell carcinoma in a 56-year-old patient. Many somatic mutations including cancer-associated gene mutations in ARID1A, ATM, CDH4, NRAS and PIK3CA were shared among epithelium samples from uterine endometrium, endometriotic lesions distant from and adjacent to the carcinoma, and the carcinoma. The mutant allele frequencies of shared mutations increased from uterine endometrium to distant endometriosis, adjacent endometriosis, and carcinoma. Although a splice site mutation of ARID1A was shared among the four epithelium samples, a frameshift insertion in ARID1A was shared by adjacent endometriosis and carcinoma samples, suggesting that the biallelic mutations triggered malignant transformation. Somatic copy number alterations, including loss of heterozygosity events at PIK3CA and ATM, were identified only in adjacent endometriosis and carcinoma, suggesting that mutant allele-specific imbalance is another key factor driving malignant transformation. By reconstructing a clonal evolution tree based on the somatic mutations, we showed that the epithelium samples were derived from a single ancestral clone. Although the study was limited to a single patient, the results from this illustrative case could suggest the possibility that epithelial cells of ovarian endometriosis and clear cell carcinoma were descendants of uterine endometrial epithelium.

XCL1 expression correlates with CD8-positive T cells infiltration and PD-L1 expression in squamous cell carcinoma arising from mature cystic teratoma of the ovary.

Molecular characteristics of carcinoma arising from mature cystic teratoma of the ovary (MCT) remain unclear due to its rarity. We analyzed RNA-sequencing data of 2322 pan-cancer [1378 squamous cell carcinomas (SCC), 6 adenosquamous carcinomas (ASC), and 938 adenocarcinomas (AC)] including six carcinomas arising from MCT (four SCCs, one ASC, and one AC). Hierarchical clustering and principal component analysis showed that gene expression profiles of carcinomas arising from MCT were different between each histological type and that gene expression profiles of SCCs arising MCT (MCT-SCCs) was apparently similar to those of lung SCCs. By epidermis-associated pathways activity based on gene set enrichment analysis, 1030 SCCs were divided into two groups: epidermis-signature high (head and neck, esophagus, and skin) and low (cervix, lung, and MCT). In addition to pan-SCC transcriptome analysis, cytokeratin profiling based on immunohistochemistry in the independent samples of 21 MCT-SCCs clarified that MCT-SCC dominantly expressed CK18, suggesting the origin of MCT-SCC was columnar epithelium. Subsequently, we investigated differentially expressed genes in MCT-SCCs compared with different SCCs and identified XCL1 was specifically overexpressed in MCT-SCCs. Through immunohistochemistry analysis, we identified XCL1 expression on tumor cells in 13/24 (54%) of MCT-SCCs but not in MCTs. XCL1 expression was also significantly associated with the number of tumor-infiltrating CD8-positive T cells and PD-L1 expression on tumor cells. XCL1 produced by tumor cells may induce PD1/PD-L1 interaction and dysfunction of CD8-positive T cells in tumor microenvironment. XCL1 expression may be a novel biomarker for malignant transformation of MCT into SCC and a biomarker candidate for therapeutic response to an anti-PD1/PD-L1 therapy.