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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.

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.

KRAS is the most frequently mutated in ovarian endometriosis. However, it is unclear whether the KRAS mutant allele's mRNA is expressed and plays a biological role in ovarian endometriosis. Here, we performed mutation‐specific RNA in situ hybridization to evaluate mutant allele expression of KRAS p.G12V, the most frequently detected mutation in ovarian endometriosis in our previous study, in formalin‐fixed paraffin‐embedded tissue (FFPE) samples of ovarian endometriosis, cancer cell lines, and ovarian cancers. First, we verified that mutant or wild‐type allele of KRAS were expressed in all 5 cancer cell lines and 9 ovarian cancer cases corresponding to the mutation status. Next, we applied this assay to 26 ovarian endometriosis cases, and observed mutant allele expression of KRAS p.G12V in 10 cases. Mutant or wild‐type allele of KRAS were expressed in line with mutation status in 12 available endometriosis cases for which KRAS gene sequence was determined. Comparison of clinical features between ovarian endometriosis with KRAS p.G12V mutant allele expression and with KRAS wild‐type showed that KRAS p.G12V mutant allele expression was significantly associated with inflammation in ovarian endometriosis. Finally, we assessed the spatial distribution of KRAS mutant allele expression in 5 endometriosis cases by performing multiregional sampling. Intratumor heterogeneity of KRAS mutant allele expression was observed in two endometriosis cases, whereas the spatial distribution of KRAS p.G12V mutation signals were diffuse and homogenous in ovarian cancer. In conclusion, evaluation of oncogene mutant expression will be useful for clarifying the biological significance of oncogene mutations in benign tumors., We could visualize mutant allele expression of KRAS p.G12V in ovarian endometriosis epithelial cells harboring KRAS p.G12V mutation by using the in‐situ hybridization assay and revealed biological significance of KRAS mutant allele expression in ovarian endometriosis.

The naughty genes and 3D structure of the endometrium Takayuki Enomoto, Manako Ymaguchi, Kazuaki Suda, Kosuke Yoshihara Department of Obstetrics and Gynecology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan. The human endometrium is a highly regenerative tissue and involved in menstruation and implantation of the fertilized egg, giving it a central role in women’s reproductive health. However, the regenerative nature of the endometrial glands can lead to the development and progression of “endometrium-related diseases” such as adenomyosis, endometriosis, endometriosis-associated ovarian cancer, endometrial hyperplasia, and endometrial cancer. To clarify the pathogenesis of endometrium-related diseases and develop effective preventative measures and therapeutic strategies, comprehensive understanding of molecular biological linkage between endometrium and endometrium-related diseases was crucially important. To this end, we focused on genomic alterations of endometrial epithelium which is considered the origin of endometriosis, and sequenced 107 ovarian endometriotic and 82 normal uterine endometrial epithelium samples isolated by laser-microdissection. Intriguingly, several genes recurrently mutated in endometriosis-associated ovarian cancers were frequently mutated in both endometriotic epithelium and normal uterine endometrial glands. In particular, PIK3CA mutation was detected in 41% of endometrial epithelium subjects but none of them had shared PIK3CA mutations across multiple regions collected from the same individuals. Mutation allele frequencies of somatic mutations in uterine endometrial epithelium samples were also significantly lower than those in ovarian endometriotic epithelium samples, suggesting the heterogeneous genomic compositions in uterine endometrium. To interpret this genomic heterogeneity in uterine endometrium, we focused on endometrial gland, the minimum functional unit of uterine endometrium, and conducted 109 single endometrial glands sequencing. As a result, we unveiled that each gland carried distinct somatic mutations in cancer-associated genes, such as PIK3CA, KRAS, and PTEN, with high mutant allele frequencies, suggesting the monoclonality of each gland. The presence of cancer-associated gene mutations in histologically normal endometrial glands provides important clues regarding the pathogenesis of endometrium-related diseases. However, our previous study could not determine the spread of endometrial gland harboring cancer-associated gene mutation because there is a limitation to two-dimensional assessment of the whole shapes of endometrial gland due to its complicatedly winding morphology. Therefore, we tackled with three-dimensional (3D) assessment of human endometrium. To construct a large picture of endometrial gland structure, we performed tissue-clearing-based 3D imaging of full-thickness human uterine endometrial tissue with the use of light-sheet fluorescence microscopy. Our 3D immunohistochemistry discovered some new and unique 3D morphologies of endometrial glands, including plexus network of glands or occluded glands. Notably, computational analysis of 3D layer clarified that the plexus structure of the glands was mainly located in the stratum basalis and expanded along muscular layer horizontally, similar to the so-called “rhizome of grass”. Although previous studies have shown the 3D structure of murine endometrial glands, the bottom of these glands forms a crypt but not a rhizome. This can potentially be explained by the existence of menstruation, which is the crucial difference between the human and murine endometrium. The rhizome structure of endometrial gland in the human endometrium will have a functional advantage over the crypt in terms of the conservation of progenitor/stem cells and regeneration. In addition, some endometrial glands shared the plexus and rose toward the luminal epithelium, suggesting that these glands were the same origin. The rhizome of the endometrium may be a crucial element for understanding the expansion of endometrial glands harboring cancer-associated gene mutations. Integrated analysis of the naughty gene alterations and the 3D structure in human endometrium will lead to a better understanding of the human endometrium in various fields, including histology, pathology, pathophysiology, reproduction, and oncology.

STUDY QUESTIONAre there common mutation profiles between epithelial and stromal cells in ovarian endometriotic tissue and the normal endometrium?SUMMARY ANSWEROur study revealed no common mutations between epithelial and stromal cells in ovarian endometriotic tissue and the normal endometrium.WHAT IS KNOWN ALREADYEpithelial cells in both ovarian endometriotic tissue and the normal endometrium harbor somatic mutations in cancer-associated genes such as phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) and KRAS proto-oncogene, GTPase (KRAS).STUDY DESIGN, SIZE, DURATIONWe performed a retrospective study to identify the mutation profiles of stromal cells in endometriotic tissue and the normal endometrium. We collected 11 endometriotic stroma samples and 10 normal endometrial stroma samples between 2013 and 2017 at a tertiary care center.PARTICIPANTS/MATERIALS, SETTING, METHODSThe laser microdissection method was used to obtain stromal cells in ovarian endometriotic and normal endometrial tissues from patients with ovarian endometriosis and/or other non-invasive gynecological diseases. Target gene sequencing was performed to assess and compare the mutation profiles of stromal cells with those of epithelial cells obtained in our previous study. For target gene sequencing, 76 genes were selected based on previous genomic analyses for ovarian endometriosis, normal endometrium, endometriosis-related ovarian cancer and endometrial cancer.MAIN RESULTS AND THE ROLE OF CHANCEStromal samples in ovarian endometrioma and normal endometrium harbor somatic mutations (18 mutations in 11 endometriosis samples and 16 mutations in 10 normal endometrial samples) but did not share any mutations with paired epithelial samples. The mutant allele frequency of stromal samples was significantly lower than that of epithelial samples in ovarian endometrioma (P = 6.0 × 10-11) and normal endometrium (P = 1.4 × 10-7).LIMITATIONS, REASONS FOR CAUTIONThe number of genes evaluated in the mutational analysis was limited. Additionally, the functional roles of somatic mutations in stromal cells remain unclear.WIDER IMPLICATIONS OF THE FINDINGSDifferent mutation profiles between paired epithelial and stromal cells in both ovarian endometrioma and normal endometrium suggest that origins of epithelial and stromal cells would be independent of each other in both normal endometrium and ovarian endometrioma; however, the theory of epithelial-mesenchymal transition is proposed in ovarian endometrioma.STUDY FUNDING/COMPETING INTEREST(S)This work was supported in part by the Japan Society for the Promotion of Science KAKENHI grant number JP15H02373 (Grant-in-Aid for Scientific Research A for I.I.), JP16H06267 (Grant-in-Aid for Young Scientists A for K.Y.), JP17K08688 (Grant-in-Aid for Scientific Research C for H.N.) and JP16H06279 (Grant-in-Aid for Scientific Research on Innovative Areas—Platforms for Advanced Technologies and Research Resources for H.N. and K.Y). There are no conflicts of interest to declare.TRIAL REGISTRATION NUMBERNot applicable.

Summary Advanced-stage gynecologic cancer remains a life-threatening disease. Here, we present a protocol for establishment of stable in vitro 3D spheroid cells derived from human uterine endometrial and ovarian cancer tissues. The tumor-derived spheroid cells have cancer stem cell-related characteristics, including tumorigenesis, and can be used for biological and biochemical analyses and drug efficacy assays. Because these cells possess the biological characteristics of original human tumors, spheroid cells and spheroid-derived xenografts will have applications in personalized medicine in the future. For complete details on the use and execution of this protocol, please refer to Ishiguro et al. (2016) and Mori et al. (2019)., Graphical Abstract, Highlights • Protocol for establishing spheroid cells from human gynecologic cancer tissue • A procedure to culture spheroids with cancer stem cell-related characteristics • Tumor-derived spheroids possess original human tumors’ biological characteristics, Advanced-stage gynecologic cancer remains a life-threatening disease. Here, we present a protocol for establishment of stable in vitro 3D spheroid cells derived from human uterine endometrial and ovarian cancer tissues. The tumor-derived spheroid cells have cancer stem cell-related characteristics, including tumorigenesis, and can be used for biological and biochemical analyses and drug efficacy assays. Because these cells possess the biological characteristics of original human tumors, spheroid cells and spheroid-derived xenografts will have applications in personalized medicine in the future.

Summary The fundamental morphology of the endometrial glands is not sufficiently understood by 2D observation because these glands have complicated winding and branching patterns. To construct a large picture of the endometrial gland structure, we performed tissue-clearing-based 3D imaging of human uterine endometrial tissue. Our 3D immunohistochemistry and layer analyses revealed that the endometrial glands form a plexus network in the stratum basalis and expand horizontally along the muscular layer, similar to the rhizome of grass. We then extended our method to assess the 3D morphology of tissue affected by adenomyosis, a representative “endometrium-related disease,” and observed its 3D morphological features, including the direct invasion of endometrial glands into the myometrium and an ant colony-like network of ectopic endometrial glands within the myometrium. Thus, further understanding of the morphology of the human endometrium based on 3D analysis will lead to the identification of the pathogenesis of endometrium-related diseases., Graphical abstract, Highlights • Tissue-clearing-based 3D imaging of human endometrium and adenomyosis tissue • Baseline for the 3D structure of human endometrial glands has been established • Human endometrial glands form rhizome network in the stratum basalis • Adenomyotic glands structure an ant colony-like network in uterine myometrium, Human Physiology; Imaging Anatomy

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.

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., Suda et al performed whole‐exome sequencing for normal endometrial endometrium, two ovarian endometriotic epitheliums that were distant from or contiguous to the carcinoma, and carcinoma. A substantial number of somatic mutations were shared among four epithelium samples, and the mutant allele frequencies of shared mutations increased sequentially from uterine endometrium to carcinoma. Clonal evolution analysis revealed that epithelial cells of ovarian endometriosis and clear cell carcinoma were descendants of uterine endometrial epithelium.

Sex-determining region Y-box 2 (SOX2) is an essential factor involved in the self-renewal and pluripotency of embryonic stem cells and has functions in cell survival and progression in many types of cancers. Here, we found that several endometrial cancer cell lines expressed SOX2, which was required for cell growth. Additionally, SOX2 overexpression regulated the expression of cyclin-dependent kinase inhibitor 1A (CDKN1A), and SOX2 specifically bound to p21 promoter DNA in endometrial cancer cell lines expressing SOX2. Expressions of SOX2 in endometrial cancer patients were significantly correlated with histological grade and poor prognosis. Moreover, low p21 together with high SOX2 expressions in advanced endometrial cancer patients were associated with the most unfavorable outcomes of patients. These results indicated that simultaneous measurement of SOX2 and p21 expression in endometrial cancer patients may be a useful biomarker for patient prognosis.

Intraplacental choriocarcinoma is one of the rarest forms of gestational tumors and is believed to be one of the causes of fetomaternal transfusion (FMT). A 35-year-old woman, gravida 2, para 2, with a history of two vaginal deliveries, was incidentally diagnosed as having stage I gestational intraplacental choriocarcinoma with a FIGO/World Health Organization 2000 risk score of 2 after term delivery. This disease caused neonatal anemia but did not metastasize to either the mother or infant. Short tandem repeat analysis with laser microdissection revealed that the tumor had originated from the current pregnancy. Serological test and immunohistochemical analysis revealed that the patient and her baby suffered from FMT. She has been free from disease without any medical intervention for the last 1 year. A combination of multiple biochemical analyses might help us diagnose the precursor pregnancy of a gestational choriocarcinoma and FMT.

We explored the frequency of germline and somatic mutations in homologous recombination (HR)-associated genes in major histological types of ovarian cancer. We performed targeted sequencing to assess germline and somatic mutations of 16 HR-associated genes and 4 mismatch repair (MMR) genes among 207 ovarian cancer patients (50 high-grade serous carcinomas (HGSC), 99 clear cell carcinomas (CCC), 39 endometrioid carcinomas (EC), 13 mucinous carcinomas (MC), and 6 low-grade serous carcinomas (LGSC)). Germline or somatic mutations of HR-associated genes were detected in 44% of HGSC, 28% of CCC, 23% of EC, 16% of MC, and 17% of LGSC patients. The profile of HR-associated gene mutations was remarkably different among each histological type. Germline BRCA1/2 mutations were frequently detected in HGSC and were rarely observed in CCC, EC, and MC patients. ATM somatic mutation was more frequently detected in CCC (9%) and EC patients (18%) than in HGSC patients (4%). There was a positive correlation between MMR gene mutations and HR-associated gene mutations (p = 0.0072). Our findings might be useful in selection of ovarian cancer patients that should be treated with PARP inhibitors.

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.

We previously found that therapeutic targetable fusions are detected across various cancers. To identify therapeutic targetable fusion in uterine cervical cancer, for which no effective gene targeted therapy has yet been clinically applied, we analyzed RNA sequencing data from 306 cervical cancer samples. We detected 445 high confidence fusion transcripts and identified four samples that harbored FGFR3-TACC3 fusion as an attractive therapeutic target. The frequency of FGFR3-TACC3-fusion-positive cervical cancer is also 1.9% (2/103) in an independent cohort. Continuous expression of the FGFR3-TACC3 fusion transcript and protein induced anchorage-independent growth in the cervical epithelial cell line established from the ectocervix (Ect1/E6E7) but not in that from endocervix (End1/E6E7). Injection of FGFR3-TACC3 fusion-transfected-Ect1/E6E7 cells subcutaneously into NOG mice generated squamous cell carcinoma xenograft tumors, suggesting the association between FGFR3-TACC3 fusion and squamous cell carcinogenesis. Transfection of a FGFR3-TACC3 fusion transcript into four cervical cancer cell lines (SiHa, ME180, HeLa, and Ca Ski) induced activation of the MAPK pathway and enhancement of cell proliferation. Transcriptome analysis of the FGFR3-TACC3 fusion-transfected cell lines revealed that an IL8-triggered inflammatory response was increased, via activation of FGFR3–MAPK signaling. Continuous expression of FGFR3-TACC3 fusion led to activation of the PI3K–AKT pathway only in the two cell lines that harbored PIK3CA mutations. Sensitivity to the FGFR inhibitor, BGJ398, was found to depend on PIK3CA mutation status. Dual inhibition of both FGFR and AKT showed an obvious synergistic effect in cell lines that harbor mutant PIK3CA. Additionally, TACC3 inhibitor, KHS101, suppressed FGFR3-TACC3 fusion protein expression and showed antitumor effect against FGFR3-TACC3 fusion-transfected cell lines. FGFR3-TACC3 fusion-positive cancer has frequent genetic alterations of the PI3K/AKT pathway and selection of appropriate treatment based on PI3K/AKT pathway status should be required.

Recent advances in RNA-sequencing technology have enabled the discovery of gene fusion transcripts in the transcriptome of cancer cells. However, it remains difficult to differentiate the therapeutically targetable fusions from passenger events. We have analyzed RNA-sequencing data and DNA copy number data from 25 endometrial cancer cell lines to identify potential therapeutically targetable fusion transcripts and have identified 124 high-confidence fusion transcripts, of which 69% are associated with gene amplifications. As targetable fusion candidates, we focused on three in-frame kinase fusion transcripts that retain a kinase domain (CPQ-PRKDC, CAPZA2-MET and VGLL4-PRKG1). We detected only CPQ-PRKDC fusion transcript in three of 122 primary endometrial cancer tissues. Cell proliferation of the fusion-positive cell line was inhibited by knocking down the expression of wild-type PRKDC but not by blocking the CPQ-PRKDC fusion transcript expression. Quantitative real-time RT-PCR demonstrated that the expression of the CPQ-PRKDC fusion transcript was significantly lower than that of wild-type PRKDC, corresponding to a low transcript allele fraction of this fusion, based on RNA-sequencing read counts. In endometrial cancers, the CPQ-PRKDC fusion transcript may be a passenger aberration related to gene amplification. Our findings suggest that transcript allele fraction is a useful predictor to find bona-fide therapeutic-targetable fusion transcripts.

Uterine cervical cancer is one of the most common cancer in women worldwide, and the prognosis in advanced or recurrent cases remains poor because of no effective molecular target therapies for this disease. The aim of our study is to identify and validate therapeutically targetable gene fusions in uterine cervical cancer, leading to the development of new therapeutic strategies. We have analyzed RNA sequencing data of 253 TCGA cervical cancer samples to search for gene fusions by using PRADA algorithm, and validated recurrent fusions in our Japanese dataset (n = 100) by RT-PCR and sanger sequencing. In TCGA samples, we detected 358 fusion transcripts and extracted 3 kinds of recurrent in-frame fusion transcripts (FGFR3-TACC3, ARL8B-ITPR1 and NXN-ABR). We focused on FGFR3-TACC3 fusion as a candidate of therapeutic kinase fusion and identified FGFR3-TACC3 fusion in 2 of 253 (0.8%) TCGA samples and 2 of 100 (2%) Japanese samples. All of fusion positive samples, which were histologically diagnosed as squamous cell carcinoma, showed high expression of both FGFR3 and TACC3 genes. To evaluate the possibility as therapeutically targetable fusions in uterine cervical cancer, we transfected FGFR3-TACC3, wild type FGFR3 and TACC3 transcripts to normal immortalized cervical keratinocytes derived from ectocervix (Ect1/E6E7) and four uterine cervical cancer cell lines (SiHa, ME180, HeLa, CaSki). Continuous expression of FGFR3-TACC3 fusion transcript led to anchorage-independent growth in Ect1/E6E7 and the dramatically alteration of cell proliferation in all cancer cell lines. On the other hand, no obvious phenotype change was observed in FGFR3 or TACC3 transfected cells. Western blotting analysis demonstrated that MAPK pathway was activated in all the fusion transfected cell lines but PI3K/AKT pathway was activated only in ME180 and CaSki harboring PIK3CA mutation. Fusion transfected cell lines exhibited high sensitivity to fusion specific siRNA and FGFR inhibitor compared to control. Although PI3K/AKT activated cell lines (ME180 and CaSki) showed less effective against FGFR inhibitor compared to PI3K/AKT neutral cell lines (SiHa and HeLa), combined inhibition of FGFR and AKT had a synergic effect in PI3K/AKT activated cell lines. Our findings suggest FGFR3-TACC3 fusion gene is an oncogenic driver event and therapeutic target in a fraction of cervical cancer. When FGFR3-TACC3 fusion positive cervical cancer is treated by FGFR inhibitor, genomic background such as PI3K/AKT status should be considered. Citation Format: Ryo Tamura, Kosuke Yoshihara, Tetsuya Saito, Ryosuke Ishimura, Emmanuel Martínez-Ledesma, Yutaro Mori, Kaoru Yamawaki, Kazuaki Suda, Tatsuya Ishiguro, Yoichi Aoki, Seiya Sato, Hiroaki Itamochi, Masaaki Komatsu, Roeland Verhaak, Takayuki Enomoto. The significance of activated PI3K/AKT pathway in FGFR3-TACC3 fusion positive cervical cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 529. doi:10.1158/1538-7445.AM2017-529

The prognosis of patients with advanced stage or recurrent endometrial cancer remains poor and there is at present no effective therapeutic molecular target for this disease. Our aim is to identify therapeutic targetable targets in endometrial cancer. We focused on fusion transcripts whose inhibitors show impressive activity in some tumors and have analyzed RNA-sequencing data and DNA copy number data from 25 endometrial cancer cell lines to identify potential therapeutically targetable fusion transcripts. We have identified 124 high-confidence fusion transcripts, of which 69% are associated with gene amplifications but have not detected recurrent fusion transcripts. We performed Sanger sequencing of RT-PCR amplification products and validated 27 (93.1%) of the 29 fusion transcripts that were randomly selected. As targetable fusion candidates, we extracted three in-frame kinase fusion transcripts that retain a kinase domain (CPQ-PRKDC, CAPZA2-MET, and VGLL4-PRKG1). We detected only CPQ-PRKDC fusion transcript in three of 122 primary endometrial cancer tissues. Cell proliferation of the fusion-positive cell line was inhibited by knocking down the expression of wild-type PRKDC but not by blocking the CPQ-PRKDC fusion transcript expression. Quantitative real-time RT-PCR demonstrated that the expression of the CPQ-PRKDC fusion transcript was significantly lower than that of wild-type PRKDC, corresponding to a low transcript allele fraction of this fusion, based on RNA-sequencing read counts. Western blotting did not show the detectable protein level of the CPQ-PRKDC similar to CAPZA2-MET, and VGLL4-PRKG1. In endometrial cancers, the CPQ-PRKDC fusion transcript may be a passenger aberration related to gene amplification. In conclusion, we have described the landscape of fusion transcripts in endometrial cancer by using RNA-sequencing data. We could not identify therapeutically targetable kinase fusions in a small set of 25 endometrial cancer cell lines. Our findings suggest that the transcript allele fraction is a good predictor for determining bona fide targetable fusion transcripts when applying fusion detection methods that use RNA-sequencing data. Citation Format: Ryo Tamura, Kosuke Yoshihara, Kazuaki Suda, Kaoru Yamawaki, Tatsuya Ishiguro, Sosuke Adachi, Takayuki Enomoto. Identification of novel kinase fusion transcripts in endometrial cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1504.

High-grade serous ovarian cancer comprises approximately 40% of epithelial ovarian cancer cases and is the most aggressive histologic type. Because this type of cancer usually presents as advanced stage disease at the time of diagnosis, the clinical outcome in patients with advanced stage ovarian cancer remains poor. A recent study has identified that homozygous deleted essential-redundant genes generate therapeutic vulnerabilities in glioblastoma multiforme. On the other hand, a pan-cancer analysis of somatic copy number alterations demonstrates that ovarian cancer has high frequent somatic copy number alterations compared to other tumor types including glioblastoma. Therefore, we hypothesized that ovarian cancer has also therapeutic vulnerabilities leading to development of new therapeutic strategies. To discover ovarian cancer specific vulnerability, we used DNA copy number and mRNA expression data submitted to the Cancer Genome Atlas. First, we performed GISTIC 2.0 to detect copy number alterations and found 4107 genes with homozygous deletion in at least 1% of 562 ovarian cancer samples. Of them, 3060 genes had at least one paralogue gene defined by Ensembl. We focused on metabolism pathway to extract 182 metabolism-related genes. By comparing gene expression between homozygous deletion samples and others using unpaired t-test, we identified 73 genes that were significantly down-regulated in homozygous deletion samples (p < 0.05). Next, we used copy number data submitted to Cancer Cell Line Encyclopedia to discover 41 candidate genes that were homozygously deleted in at least one ovarian cancer cell line. These findings suggest that cancer specific vulnerability exists not only glioblastoma multiforme but also other tumor types including ovaryan cancer and that further functional validation of 41 candidate genes might lead to development of new therapeutic strategies in ovarian cancer in the near future. Citation Format: Kosuke Yoshihara, Ryo Tamura, Kazuaki Suda, Tatsuya Ishiguro, Takayuki Enomoto. Ovarian cancer specific therapeutic vulnerability. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 99.