Search

Single cell RNA sequencing (scRNA-seq) has gained increased popularity in recent years and has revolutionized the study of cell populations; however, this technology presents several caveats regarding specific gene expression measurement. Here we examine the expression levels of several immune checkpoint genes, which are currently assessed in clinical studies. We find that unlike in most bulk sequencing studies, PVRIG, a novel immune-modulatory receptor in the DNAM-1 axis, suffers from poor detection in 10x Chromium scRNA-seq and other types of assays that utilize the GENCODE transcriptomic reference (gene model). We show that the default GENCODE gene model, typically used in the analysis of such data, is incorrect in the PVRIG genomic region and demonstrate that fixing the gene model recovers genuine PVRIG expression levels. We explore computational strategies for resolving multi-gene mapped reads, such as those implemented in RSEM and STARsolo and find that they provide a partial solution to the problem. Our study provides means to better interrogate the expression of PVRIG in scRNA-seq and emphasizes the importance of optimizing gene models and alignment algorithms to enable accurate gene expression measurement in scRNA-seq and bulk sequencing. The methodology applied here for PVRIG can be applied to other genes with similar issues.

According to the 2004 Japanese definition, early-onset (EO) preeclampsia (PE) is defined as PE occurring at32 weeks of gestation. This was based on the presence of "dual peaks" (30-31 and 34-35 weeks) in the prevalence of severe forms of hypertension. In contrast, the international definition adopted a cutoff of 34 weeks based on the consensus. Our aim was to investigate whether there were "dual peaks" in the gestational-age-specific incidence or prevalence of PE onset in pregnant women who underwent maternal check-ups at20 weeks of gestation in a multicenter retrospective cohort study. Diagnoses of PE and superimposed preeclampsia (SPE) were based on the new Japanese definition. A total of 26,567 pregnant women with singleton pregnancy were investigated. The best fitting equations for the distribution of the onset of gestational-age-specific incidence (hazard) rates of PE/SPE, PE, and PE with severe hypertension (a systolic blood pressure ≥160 and/or a diastolic blood pressure ≥110 mmHg) were investigated using the curve estimation function in SPSS. PE/SPE occurred in 1.83% of the patients. EO-PE/SPE with onset at32 and34 weeks of gestation and preterm PE/SPE occurred in 0.38, 0.56, and 1.07% of the patients, respectively. Gestational-age-specific incidence rates of PE/SPE, PE, and PE with severe hypertension showed exponential increases, with very high R

Ovarian cancer (OC) is the fifth leading cause of cancer death in women in the United States. Standard of care (surgical resection and platinum-based chemotherapy) has limited survival benefit for patients with advanced disease. Since 3 of every 4 patients are diagnosed with stage III-IV disease, novel therapeutic approaches are needed to improve survival. In trials with PD1/PD-L1 blockers, patients with OC had modest responses, highlighting an unmet need for effective immunotherapies. Our group has shown the PVRL2-PVRIG node in the DNAM-1 axis suppresses CD8 T cell effector function in syngeneic tumors as well as patient-derived ovarian tumors. This study aims to determine the requirement of tumor- vs myeloid cell-derived PVRL2 for driving immune suppression in OC. Taqman qPCR was conducted for PVRL2, PVR and PD-L1 mRNA expressions of classically activated (M1), and alternatively activated (M2) macrophages from wild-type and PVRL2−/− mice. In our syngeneic OC model, ID8-Vegf-Defb29 (50,000 cells/ovary) were surgically implanted in the ovarian bursa and monitored for disease progression. These studies were performed in (1) PVRL2−/− mice vs. wild type mice and (2) PVRL2−/− → Wild-type vs. wild-type → wild-type bone marrow chimeras. PVRL2−/- ID8-Vegf-Defb29 clones were generated by CRISPR-Cas9, validated for absence of off-target edits by whole exome sequencing and implanted in wild-type and PVRL2−/− mice. Multiplex immunofluorescence (mIF) of PanCK/CD8-CD68-PVRL2/PVR was performed on archived HGSOC tissue from patients treated at Johns Hopkins Hospital. CITE-seq was performed on viable TILs from 6 treatment-naïve and 4 neoadjuvant chemotherapy-treated patients. PVR transcripts were elevated in PVRL2−/− M1 (3.77-fold, p=0.04) and M2 (2.61-fold, p=0.003) macrophages compared to wild type. The expression of PD-L1 was increased in M2 macrophages of PVRL2-/- mice compared to wild-type mice (Mean ratio: 2.28, p=0.04). PVRL2−/− mice and PVRL2 bone marrow chimeras had significantly improved survival compared to their respective control cohorts. Tumors derived from PVRL2−/− ID8-Vegf-Defb29 clones grew slowly in PVRL2−/− mice compared to wild-type mice. PVRL2 and PVR were highly expressed in human HGSOC, and the prognosis was worse for patients with high PVRL2 expression. In mIF, PVRL2 was expressed not only on tumor cells but also on CD68-positive cells. In CITE-seq, PVRL2 and PVR were strongly expressed in macrophage and dendritic cell clusters. The enhanced expression of PVR and PD-L1 in PVRL2−/− M1 and M2 macrophages suggests compensatory activation of co-inhibitory signaling pathways. PVRL2 deficiency selectively in hematopoietically-reconstituted immune cells (primarily myeloid cells) and not in tumor cells conferred a significant survival benefit in our syngeneic OC model. Taken together, blockade of the PVRL2-PVRIG axis in conjunction with PD-1/L1 inhibition may be an effective modality in OC. Citation Format: Kosuke Murakami, Hirofumi Ando, Michele Doucet, William Huang, Sudipto Ganguly. The role of myeloid cell-expressed PVRL2 in high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6374.

Single cell RNA sequencing (scRNA-seq) has gained increased popularity in recent years and has revolutionized the study of cell populations; however, this technology presents several caveats regarding specific gene expression measurement. Here we examine the expression levels of several immune checkpoint genes, which are currently assessed in clinical studies. We find that unlike in most bulk sequencing studies, PVRIG and murine TIGIT suffers from poor detection in 10x Chromium scRNA-seq and other types of assays that utilize the GENCODE gene model. We show that the default GENCODE gene model, typically used in the analysis of such data, is incorrect in the PVRIG genomic region and contains also a predicted read-through transcript to which PVRIG reads co-align, causing these to be discarded and hence hindering its proper detection. Moreover, we find that the murine TIGIT 3’ UTR is mis-annotated, leading to the loss of legitimate reads. We thus generated a corrected reference genome, by removing the faulty read-through in the case of PVRIG and by extending the TIGIT 3’ UTR and demonstrate that by employing these changes we can correctly capture genuine expression levels of these checkpoints, and which align with our findings at the protein level using FACS and CITEseq. Furthermore, we show that specialized read multimap algorithms such as RSEM and STARsolo can also partially improve the detection of PVRIG. Our study provides means to better interrogate the expression of PVRIG and murine TIGIT in scRNA-seq and emphasize the importance of optimizing gene models and alignment algorithms to enable accurate gene expression measurement in scRNA-seq and bulk sequencing. Moreover, our results support detailed study of the expression of immune checkpoints in clinical and pre-clinical studies towards the development of cancer immunotherapy treatments. Citation Format: Sergey Nemzer, Niv Sabath, Assaf Wool, Zoya Altber, Hirofumi Ando, Drew M. Pardoll, Sudipto Ganguly, Yaron Turpaz, Zurit Levine, Roy Z. Granit. Gene model correction for PVRIG and TIGIT in single cell sequencing data enables accurate detection and study of its functional relevance. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4292.

Serous carcinoma (SC) is an aggressive histologic type of endometrial carcinoma (EMC) with a poor prognosis. The development of novel therapeutics for SC is an important issue. PIM1 is a serine/threonine kinase involved in various cellular functions, such as cell cycle progression, apoptosis, and transcriptional activation via the phosphorylation of many target proteins, including MYC. PIM1 is overexpressed in several cancers and has been associated with the treatment-resistance. We investigated the expression and function of PIM1 in EMC, particularly SC. Immunohistochemical analysis in 133 EMC cases [103 endometrioid carcinomas (EC) and 30 SC] revealed the significantly stronger expression of PIM1 in SC than in EC and significantly shorter survival of patients with the overexpression of PIM1 in all EMC cases, as well as in only SC cases. A multivariate analysis identified the overexpression of PIM1 as an independent prognostic factor. The knockdown of PIM1 by siRNA in the SC cell line, ARK1, decreased the expression of phosphorylated MYC and reduced proliferation, migration, and invasion. The PIM1 inhibitor, SGI-1776, reduced cell viability in SC cell lines (ARK1, ARK2, and SPAC1L) with IC50 between 1 and 5 µM. SGI-1776 also reduced the migration and invasion of ARK1 cells. Moreover, the oral administration of SGI-1776 significantly suppressed subcutaneous ARK1 xenograft tumor growth in nude mice without impairing health. These results indicate that PIM1 is involved in the acquisition of aggressiveness and suggest the potential of PIM1 as a novel therapeutic target and SGI-1776 as a therapeutic agent for SC.

IntroductionAlthough lobular endocervical glandular hyperplasia is a benign disorder of the uterine cervix, its potential as a precursor of minimal deviation adenocarcinoma has been reported. However, the natural history of the disease and the frequency of malignant change are not fully understood. We evaluated the frequency of malignant change of clinical lobular endocervical glandular hyperplasia and explored useful parameters indicating malignant change.MethodsThe clinical courses of 175 patients with cervical multi-cystic lesions who visited Shinshu University Hospital between June 1995 and June 2019 were retrospectively analyzed. We examined the results of follow-up and outcomes of the patients diagnosed with lobular endocervical glandular hyperplasia and investigated the frequency of malignant transformation.ResultsOf the 175 patients, 15, 84, and 76 were clinically diagnosed with suspected malignancy, suspected lobular endocervical glandular hyperplasia, and suspected nabothian cyst, respectively. Of these patients, 69 patients with suspected lobular endocervical glandular hyperplasia were followed, and 12 underwent hysterectomy after a mean follow-up of 57.1 (range: 3–154) months due to lesion enlargement (increase in tumor diameter of >20%) and/or worsening cytology. Of these 12 patients, two had lobular endocervical glandular hyperplasia with atypia and one had minimal deviation adenocarcinoma. Of 69 patients, the rate of malignant change was 1.4% (1/69). The growth rates of the lesions for these three patients during follow-up were significantly higher than those of nine patients who underwent surgery with lobular endocervical glandular hyperplasia without atypia and 48 follow-up cases of suspected lobular endocervical glandular hyperplasia. The cut-off value of the growth rate suggesting malignant transformation was 38.1% (84.6% sensitivity and 100% specificity). Tumor size and cytology did not change in the remaining 57 cases continuing follow-up.ConclusionAn increase in tumor size and worsening cytology are important parameters for detecting malignant transformation of lobular endocervical glandular hyperplasia during follow-up. However, the frequency of malignant change of this disease may be limited. These results suggest that conservative management may be an option for clinical lobular endocervical glandular hyperplasia.

In order to identify genes involved in the pathogenesis of clear cell carcinoma of the ovary (CCC), functional screening using a cDNA expression library was performed. We extracted mRNA from a CCC cell line (RMG-1), established a cDNA library using a retroviral vector, transfected that library into mouse NIH3T3 cells and sequenced the resultant foci. The tissue-type specific expression of isolated genes and their transforming activities were evaluated. Seven genes were isolated. Of these genes, the mRNA expression of SEC61B and DVL1 is significantly stronger in CCC than in other histological types (

Acute lymphocytic myocarditis in pregnancy is rare, with no established management guidelines to date. A 40-year-old woman at 34 weeks of gestation complained of shortness of breath upon exertion. An electrocardiogram revealed broad ST elevation, and echocardiography showed diffuse impairment of left ventricular contractility. The patient was immediately transferred to our hospital for suspected takotsubo cardiomyopathy. We considered myocarditis based on the patient’s prior cold-like symptoms and additional examination. Myocardial biopsy revealed lymphocyte infiltration, which confirmed acute lymphocytic myocarditis. Although there were no signs of heart failure or conduction disturbance under catecholamine, her hemodynamics were weak. Emergency cesarean section was performed because of possible hemodynamic failure during the remaining course of pregnancy. Both the mother and baby were discharged without any subsequent events. If acute myocarditis is suspected during pregnancy, prompt myocardial biopsy is crucial for timely pathological diagnosis and treatment decisions. Clinicians should consider premature delivery prior to a possible failure in maternal hemodynamics.

A 29-year-old woman was admitted to our hospital due to diagnosis of pregnancy at 5 weeks and a day. She underwent valve replacement with mechanical heart valve (MHV: SJM valve) for congenital mitral valve regurgitation, when 11 years old. Warfarin 4 mg was used for anticoagulation. After admission, warfarin was replaced by unfractionated heparin (UFH). She developed exertional dyspnea at 8 weeks of pregnancy. Echocardiogram and fluoroscopy showed an immobile leaflet in the closed position. She was diagnosed with mechanical valve thrombosis. Cardiac surgery or thrombolytic therapy (TT) were treatment options. TT is not established, but is reported to be safer than cardiac surgery. Recently, low-dose, slow infusion of recombinant tissue plasminogen activator (rt-PA) therapy showed acceptable results. About 2.5 h after an intravenous injection of rt-PA, diastolic rumble improved to the normal range of leaflet. Thereafter, warfarin was restarted and there was no recurrence of symptoms and no abortion. She was readmitted for the scheduled Caesarean section (CS) at 32 weeks of pregnancy, and warfarin was replaced with UFH. At 34 weeks of pregnancy, a baby was delivered by CS. She suffered hemostasis after surgery under the anticoagulation. Postoperative day 31, both mother and a child were healthy and left the hospital.

As one of the super-aged countries, Japan is facing serious social and economic implications of the rapidly increasing elderly population while the working population is decreasing. This has created tremendous burden not only on the Government of Japan but also on the local administrative units like Kobe City to provide adequate health and social welfare programme support to the elderly. Thus, this study examines the birth and the programme of the Silver College as a key component of the general support system for the elderly of Kobe City initially within the context of its overall economic development process in the 1980s–1990s. It also attempts to assess its contribution to the local communities. The qualitative research approach was employed through interviews with the founders and managers of the Silver College in Kobe City to help better understand the longitudinal and complex reality of its issues and contribution to the ageing society in the City of Kobe. Personal observations of the classes and activities of the Silver College students outside the Silver College and reviews of documents published about the Silver College helped to further understand its meaningful relationship between the students and their local communities, hence adhering to the naturalistic paradigm. The results revealed that the key critical successful factor of Kobe City elderly care system is through its unique community-related reciprocal support system, which is organised and operated with the primary aim of helping the elderly continue to live with acceptable healthy living conditions, not only physically but also mentally so as to contribute to improving their quality of life at the community level. While challenges can be increasing, the study found that the social welfare programme of the City of Kobe especially with its Silver College would continue to help the increasing number of the elderly citizens to lead a healthier life and to increase their contribution to their respective communities in the foreseeable future.

In order to understand the role of gene mutations in endometrial carcinogenesis, whole exome sequencing via laser microdissection was performed in the normal endometrium, atypical endometrial hyperplasia and endometrial carcinoma in the same patient. A total of 4046 and 5746 mutations with amino acid substitution were detected in endometrial hyperplasia and endometrial carcinoma, respectively; 2252 were common in both tissues and might play crucial roles in early carcinogenesis. These common mutations included polymerase epsilon (POLE) and DNA mismatch repair (MMR) genes, indicating that an ultra-mutated phenotype, and also included PTEN and PIK3CA. The mutation-prone environment evoked by mutations in the POLE and MMR genes associated with the activated phosphatidylinositol-3 kinase pathway played a pivotal role in this case.

Sirtuin 1 (SIRT1), originally identified as a longevity gene, regulates DNA repair and metabolism by deacetylating target proteins such as p53. SIRT1 plays a key role in the pathophysiology of metabolic diseases and neurodegenerative disorders, and is considered to protect against age-related diseases including cancer. In contrast, SIRT1 may be oncogenic because its overexpression has been detected in many cancers. The aim of the present study was to clarify the expression and the role of SIRT1 in ovarian carcinoma (OvCa). The expression of SIRT1 was evaluated immunohistochemically in 16 cases of normal ovaries, 35 cases of endometriosis with/without carcinoma, and 68 cases of OvCa (endometrioid, 16; clear cell, 20; mucinous, 16; serous, 16). Staining results were evaluated semiquantitatively by the Immunoreactive Scoring System, and the relationships with clinicopathologic features and outcomes of patients were analyzed. The expression of SIRT1 was higher in endometrioid, mucinous, and clear-cell carcinomas than in the inclusion cysts of normal ovaries, but not in serous carcinoma (P=0.038). The expression of SIRT1 on OvCa did not correlate with age, stage, location of metastasis, or capsular penetration. However, elevated SIRT1 expression was a significant predictor of shorter survival in univariate (P=0.038) and multivariate (P=0.037) survival analyses, regardless of the tumor stage. Results of the present study suggest a positive role for SIRT1 in the development of OvCa and its potential as a novel therapeutic target.

Lobular endocervical glandular hyperplasia (LEGH) was first reported as a benign proliferative disorder of the uterine cervix. However, it currently remains unclear whether it has the biological characteristics of pyloric metaplasia or precursor of minimal deviation adenocarcinoma (MDA)/gastric-type mucinous cervical adenocarcinoma (GAS). Therefore, in the present study we performed whole-exome sequencing on three cases of LEGH collected by laser-microdissection from the frozen tissue sections of surgically removed uteri. Analysis of the results identified 50 somatic variants. After several filtering processes, we identified 13 functional variants, including 12 missense and one insertion-deletion variants. Of these mutations, keratinocyte proline-rich protein, olfactory receptor M4 and zinc finger protein 645 mutations were found in the Catalogue Of Somatic Mutations In Cancer but were not related to carcinogenic diseases. We did not detect any significant copy number alterations or signatures. Although this was a limited case series, we did not identify any variants relevant to the tumorigenesis of LEGH to MDA/GAS, suggesting a metaplastic aspect of LEGH.

Background The sawtooth fetal heart rate pattern is rare, and has been reported as a possible indicator of neurological sequelae in newborns. However, we observed this fetal heart rate pattern in an infant with normal neurological function. Case presentation A 29-year-old primigravida Japanese woman presented to our hospital at 40 weeks and 1 day of gestation with marked vaginal bleeding. Since admission, fetal heart rate tracing consistently demonstrated a sawtooth-like pattern. There were 3–4 oscillations per minute, and their amplitude was 30–40 beats per minute. An emergency cesarean section was performed because of non-reassuring fetal status. Evidence of placental abruption was not observed. The newborn was a male weighing 2936 g, with an Apgar score of 1 and 3 at 1 minute and 5 minutes, respectively. The infant received brain cooling, but was discharged uneventfully. A follow-up examination at age 3 years demonstrated no developmental restriction. Conclusion Although the Apgar score of the newborn was low, the infant had no neurological sequelae. Thus, the sawtooth fetal heart rate pattern may not be linked to in utero irreversible fetal central nervous system injury.

Aim The proper preoperative diagnosis and management of cervical proliferative disorders presenting with multiple cysts, including minimal deviation adenocarcinoma (MDA), lobular endocervical glandular hyperplasia (LEGH), and nabothian cyst (NC), have not been fully established. We previously proposed a management protocol comprising a diagnostic approach using cytology, magnetic resonance imaging, and gastric-type mucin and subsequent treatment. We herein evaluate the usefulness of this protocol and implications of GNAS mutations in LEGH. Methods The clinical courses of 94 patients with cervical multicystic lesions who visited our hospital between June 1995 and September 2014 were retrospectively analyzed. GNAS mutations were investigated in 10 LEGH, five LEGH with atypia, and two MDA cases. Results Of the 94 patients, the conditions of 10, 59, and 25 were clinically diagnosed as suspicious of MDA or carcinoma (S/O MDA-Ca), suspicious of LEGH (S/O LEGH), and NC, respectively. Ten patients each with S/O MDA-Ca and S/O LEGH underwent hysterectomy, and the correct ratio for diagnosis was 90% (18/20). Of the 42 S/O LEGH cases followed-up for more than 12 months, three showed an increase in tumor size. After hysterectomy, two were LEGH with atypia while one was NC. The GNAS mutation was detected in two cases of LEGH with atypia, one of which showed an increase in tumor size during follow-up. Conclusion The management protocol we propose herein will be useful. An increase in tumor size is important to detect potentially malignant LEGH. GNAS mutations may be involved in the tumorigenesis of potentially malignant LEGH.

Sirtuin 1 (SIRT1), originally identified as a longevity gene, is induced by caloric restriction, and regulates various cellular functions including DNA repair, cell survival and metabolism via the deacetylation of target proteins such as histone and p53. These functions are considered to act dualistically as preventing or facilitating cancer. This study aimed to clarify the expression and role of SIRT1 in endometrial carcinoma. Because a high-calorie diet was a well-known risk factor for endometrial carcinoma, we first hypothesized that SIRT1 might be downregulated in normal endometrial glandular cells of obese women. However, no correlation was observed between the expression of SIRT1 and body mass index (BMI). In contrast, regardless of BMI, the immunohistochemical expression of SIRT1 was significantly higher in endometrial carcinoma (108 cases) than in normal endometria (60 cases) (P

Uterine sarcomas are neoplastic malignancies that typically arise in tissues of a mesenchymal origin in uterine body. The identifi cation of novel molecular mechanisms leading to sarcoma formation, and the establishment of new therapies and biomarkers has been hampered by several critical factors.

Human uterine leiomyosarcoma (U-LMS) is neoplastic malignancy that typically arises in tissues of mesenchymal origin. The identification of novel molecular mechanism leading to human U-LMS formation and the establishment of new therapies has been hampered by several critical points. We earlier reported that mice with a homozygous deficiency for proteasome beta subunit 9 (Psmb9)/β1i, an interferon (IFN)-γ inducible factor, spontaneously develop U-LMS. The use of research findings of the experiment with mouse model has been successful in increasing our knowledge and understanding of how alterations, in relevant oncogenic, tumour suppressive, and signaling pathways directly impact sarcomagenesis. The IFN-γ pathway is important for control of tumour growth and invasion and has been implicated in several malignant tumours. In this study, experiments with human tissues revealed a defective expression of PSMB9/β1i in human U-LMS that was traced to the IFN-γ pathway and the specific effect of somatic mutations of JANUS KINASE (JAK) 1 molecule or promoter region on the locus cording PSMB9/β1i gene. Understanding the molecular mechanisms of human U-LMS may lead to identification of new diagnostic candidates or therapeutic targets against human U-LMS.

Idiopathic interstitial pneumonias (IIPs) typically occur in the fifth and sixth decades, and thus are uncommon in the child-bearing age group. We herein report an extremely rare case of a pregnant woman with IIP accompanied by secondary pulmonary hypertension that had a successful cesarean delivery. A 36-year-old primigravida woman was referred to our Obstetrics department at 6 weeks of gestation. Seven years ago, she had a dry cough and moderate dyspnea, and was diagnosed with non-specific interstitial pneumonia (NSIP), a subtype of IIP. At 11 weeks of gestation, spirometry showed a vital capacity of 1.89 L (60% of predicted). An arterial blood gas analysis showed PaO2 of 77.2 mmHg and oxygen saturation of 95% in breathing room air at rest. An echocardiogram revealed mild pulmonary hypertension; systolic pulmonary artery blood pressure (PABP) of 44 mmHg. Fetal growth and amniotic fluid volume were normal. Our management plan was to maintain oxygen saturation at more than 95% of oxygen saturation and systolic PABP at less than 50 mmHg. Oxygen therapy was initiated at 13 weeks of gestation because of dyspnea at rest. At 28 weeks of gestation, an echocardiogram showed severe pulmonary hypertension with systolic PABP of 64 mmHg. Therefore, we performed cesarean section at 30 weeks of gestation. The newborn was a male weighing 1.452 g with Apgar scores of 6 and 7 at 1 min and 5 min, respectively. Although the patient’s post-operative course was uneventful, she was discharged on a regimen of oxygen therapy. The effects of pregnancy on IIPs remain unclear because of its extreme rarity. In the management of pregnant women with IIPs accompanied by secondary pulmonary hypertension, the maintenance of systolic PABP of less than 50 mmHg on an echocardiogram may be effective.

Objective: Although estrogen has been regarded as a risk factor for endometrial carcinoma, its concentrationdependent carcinogenetic effects remain unclear, because most endometrial carcinomas occur in post-menopausal women, whose serum estrogen levels are relatively low. We previously reported that high levels of estradiol (E2) may suppress endometrial carcinogenesis by up-regulating DNA mismatch repair (MMR) in vitro. The present study was undertaken to further examine the carcinogenetic role of estrogen at various concentrations in vivo. Methods: N-methyl-N-nitrosourea (MNU) was injected into the uterine cavity of 29 mice, and E2 was administered by pellets or orally. Uteri were removed for histological examinations 24 weeks later, and serum E2 levels were measured. The immunohistochemical expression of MMR proteins in uterine epithelia was investigated. Results: Of 29 mice, 8, 8, 8, and 5 showed atrophic, normal, hyperplastic, and carcinomatous endometria, respectively. The mean E2 levels of each group were 0.2 pg/ml, 3.8 pg/ml, 190.0 pg/ml, and 6.7 pg/ml, with significant differences. The expression of the MMR proteins was stronger in mice with elevated E2. Conclusion: Elevated E2 levels preferentially induced endometrial hyperplasia rather than carcinoma, and this may be mediated by MMR proteins. These results indicate that modest E2 is needed, whereas elevated E2 levels are not necessarily advantageous for carcinogenesis, suggesting the importance of low-chronic (un-opposed) estrogen in human endometrial carcinogenesis.

Although progestin has been used to treat endometrial hyperplasia and endometrial carcinoma (EC), its therapeutic efficacy is limited. In order to improve this, the underlining mechanisms of the effects of progestin need to be elucidated in more detail. In the present study, we examined the involvement of mitogen-inducible gene-6 (MIG6), a negative regulator of the EGF receptor, in the progestin-mediated growth suppression of endometrial epithelia. The immunohistochemical expression of MIG6 was elevated in the early to mid-secretory phases of normal endometrium and also with endometrial hyperplasia after medroxyprogesterone acetate (MPA) therapy. The addition of progesterone (P4) to progesterone receptor (PR)-positive EC cells reduced the viability and induced MIG6 messenger RNA (mRNA) and protein expression. The silencing of MIG6 using siRNA eliminated the P4-mediated reduction of EC cell viability, indicating that MIG6 is an essential downstream component of PR-mediated growth suppression. In order to enhance PR-driven signals, we examined the effects of histone deacetylase (HDAC) inhibitors because histone acetylation has been shown to increase the expression of PR. The addition of three HDAC inhibitors (panobinostat, LBH589; trichostatin A, TSA; suberoylanilide hydroxamic acid, SAHA) decreased the viability of EC cells and up-regulated the expression of PR and MIG6, and these effects were the strongest with LBH589. The addition of LBH589 and MPA synergistically decreased the viability and increased apoptosis in EC cells. These results indicate that LBH589 has potential as an enhancer of progestin therapy via the up-regulation of PR and MIG6.

Patients with uterine leiomyosarcoma (LMS) typically present with vaginal bleeding, pain, and a pelvic mass, with atypical presentations of hypercalcemia and eosinophilia also being reported. Radiographic evaluation with combined positron-emission tomography/computed tomography may assist in diagnosis and surveillance in women with uterine LMS; these are commonly used with stage and tumour grade as prognostic indicators and a recently developed risk-assessment index to predict disease-specific survival. Recent studies have shown that the addition of adjuvant therapy after surgical management does not seem to improve survival, and ovarian preservation does not appear to negatively impact outcome. Experimentally, it is noteworthy that proteasome subunit beta 9 (PSMB9)/β1i-deficient mice exhibit spontaneous development of uterine LMS, with a disease prevalence of ~37% by 12 months of age. Furthermore, a recent report showed the loss of ability to induce PSMB9/β1i expression, that is up-regulated by interferon-γ (IFNγ), in human uterine LMS tissues. Here, we reviewed human uterine LMS for genetic mutations in the IFNγ signal cascade, and found serious mutations in three genes, Janus activated kinase 1 (JAK1), signal transducer and activator of transcription 1 (STAT1) and PSMB9/β1i promoter regions. Moreover, molecular experiments demonstrated differential expression of cyclin E and P27/KIP1, that regulate cell-cycle G1 arrest via PSMB9/β1i expression. The discovery of this mutational activation of a key cell-signalling pathway may provide new targets for diagnostic approaches and therapeutic intervention.

Ovarian clear cell carcinoma (CCC) arises from ovarian endometriosis. Intra-cystic fluid contains abundant amounts of free iron, which causes persistent oxidative stress, a factor that has been suggested to induce malignant transformation. However, the mechanisms linking oxidative stress and carcinogenesis in CCC currently remain unclear. Lipocalin 2 (LCN2), a multifunctional secretory protein, functions as an iron transporter as well as an antioxidant. Therefore, we herein examined the roles of LCN2 in the regulation of intracellular iron concentrations, oxidative stress, DNA damage, and antioxidative functions using LCN2-overexpressing (ES2), and LCN2-silenced (RMG-1) CCC cell lines. The results of calcein staining indicated that the up-regulated expression of LCN2 correlated with increases in intracellular iron concentrations. However, a DCFH-DA assay and 8OHdG staining revealed that LCN2 reduced intracellular levels of reactive oxygen species and DNA damage. Furthermore, the expression of LCN2 suppressed hydrogen peroxide-induced apoptosis and prolonged cell survival, suggesting an antioxidative role for LCN2. The expression of mRNAs and proteins for various oxidative stress-catalyzing enzymes, such as heme oxygenase (HO), superoxide dismutase (SOD), and glutathione peroxidase, was not affected by LCN2, whereas the intracellular concentration of the potent antioxidant, glutathione (GSH), was increased by LCN2. Furthermore, the expression of xCT, a cystine transporter protein, and CD44 variant 8-10 (CD44v), a stem cell marker, was up-regulated by LCN2. Although LCN2 increased intracellular iron concentrations, LCN2-induced GSH may catalyze and override oxidative stress via CD44v and xCT, and subsequently enhance the survival of CCC cells in oxidative stress-rich endometriosis.

Purpose Lipocalin 2 (LCN2) is a secretory protein that is involved in various physiological processes including iron transport. We previously identified LCN2 as an up-regulated gene in endometrial carcinoma, and found that the overexpression of LCN2 and its receptor, SLC22A17, was associated with a poor prognosis. However, the functions and mechanism of action of LCN2 currently remain unclear. Methods The LCN2-overexpressing endometrial carcinoma cell lines, HHUA and RL95-2, and LCN2-low-expressing one, HEC1B, were used. The effects of LCN2 on cell migration, cell viability, and apoptosis under various stresses, including ultraviolet (UV) irradiation and cisplatin treatment, were examined using the scratch wound healing assay, WST-1 assay, and Apostrand assay, respectively. Results LCN2-silencing using shRNA method significantly reduced the migration ability of cells (p

Background: Ovarian clear cell carcinoma (OCCC) is one of the 4 major histological types of ovarian carcinoma (OVC) and is frequently arising from ovarian endometriotic cysts. OCCC is rare in the United States and western countries, but it is more frequent in Japan and may be seen in almost 25% of OVC. OCCC commonly shows chemo-resistance which makes difficult to treat OCCC. In order to develop the new therapeutics for OCCC, genetic abnormalities that lead to the development of OCCC need to be elucidated in detail. The comprehensive analysis using next-generation sequencing has rapidly revealed the accumulation of genetic abnormalities in many cancers. However, the significance of each abnormality has not been elucidated. Therefore, functional screening using a cDNA expression library was performed in order to identify genes involved in the pathogenesis of OCCC. Methods: The mRNA was extracted from an OCCC cell line, RMG-1, and a cDNA library was established using a retroviral vector. This cDNA library was transfected into mouse NIH3T3 cells, and then the transformed foci which formed multilayered growth were picked up and the inserted DNAs of those foci were detected by sequencing. The mRNA and protein expression and the transforming ability of the identified genes were confirmed by immunohistochemistry and focus-formation assay. Result: Seven genes were isolated; Dishevelled 1(DVL1), beta subunit of Sec61 (SEC61B), K-specific demethylase 4A (KDM4A), chromosome 1 open reading frame 38 (C1ORF38), splicing factor 1 (SF1), ribosomal protein S2 (RPS2), and ribosomal protein S3A (RPS3A). Of these genes, RT-PCR and immunostaining revealed that the expression of SEC61B and C1ORF38 was stronger than that in other histological types of ovarian cancer. A mutational analysis of the DVL1 c-terminal region detected 3 point-mutations in RMG1 cells, whereas no mutation was found in 24 cases of OCCC. Focus formation was confirmed by the transfection of SEC61B, C1ORF38, and DVL1 into NIH3T3 cells. Conclusion: These results suggest that SEC61B, C1ORF38, and DVL1 may be involved in the pathogenesis of OCCC. Citation Format: Tsutomu Miyamoto, Yasushi Yamada, Koichi Ida, Ryoichi Asaka, Hisanori Kobara, Hirofumi Ando, Hiroyasu Kashima, Akihisa Suzuki, Tanri Shiozawa. Functional screening of genes involved in carcinogenesis using a cDNA expression library of ovarian clear cell carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3091.

Background: Although estrogen has been regarded as a risk factor for endometrial carcinoma, its concentration-dependent carcinogenetic effects remain unclear, because most endometrial carcinomas occur in post-menopausal women, whose serum estrogen levels are relatively low. Because impaired mismatch repair (MMR) was reportedly crucial in the early stages of endometrial carcinogenesis, we focused on the relationship between various estrogen concentration and MMR activity in normal and malignant endometrial glandular cells. In those cells, we reported that the protein and mRNA expression of both hMLH1 and hMSH2, the key factors of MMR, were up-regulated by estrogen stimulation. In addition, we demonstrated that the estrogen stimulation enhanced the MMR activity in those cells using in vitro MMR assay with the M13mp2 phage heteroduplex DNAs and that the proliferating cells without hMLH1/hMSH2 expressions implying “high risk” cells were more frequently observed under relative-low estrogen concentrations. In this study, to further confirm the role of E2 in vivo, we employed a mouse endometrial carcinoma model induced by intra-uterine injection of a carcinogen, N-methyl-N-nitrosourea (MNU) under various E2 concentrations. Methods: MNU was injected into the uterine cavity of 29 CD-1 mice at 6 weeks of age, and E2 was administered by pellets or orally. Uteri were removed for histological examinations 24 weeks later, and serum E2 levels were measured. The immunohistochemical expression of MMR proteins in uterine epithelia was investigated. Results: Of 29 mice, 8, 8, 8, and 5 showed atrophic, normal, hyperplastic, and carcinomatous endometria, respectively. The mean E2 levels of each group were 0.2pg/ml, 3.8pg/ml, 190.0pg/ml, and 6.7pg/ml, with significant differences. The expression of the MMR proteins was stronger in mice with elevated E2. Conclusions: Elevated E2 levels preferentially induced endometrial hyperplasia rather than carcinoma, and this may be mediated by MMR proteins. These results indicate that modest E2 is needed, whereas elevated E2 levels are not necessarily advantageous for carcinogenesis, suggesting the importance of low-chronic (un-opposed) estrogen in human endometrial carcinogenesis. Citation Format: Ryoichi Asaka, Tsutomu Miyamoto, Koichi Ida, Hodaka Takeuchi, Hirofumi Ando, Yasushi Yamada, David Hamisi Mvunta, Hisanori Kobara, Hiroyasu Kashima, Tanri Shiozawa. Suppressive role of elevated estrogen on endometrial carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5227.

PhoDiFLE est un corpus créé pour permettre la comparaison entre des productions phonétiques de français natifs et d’apprenants, à des fins didactiques. Nous présentons ici les questionnements qui ont guidé notre réflexion dans la réalisation de ce corpus. À travers une démarche mixte (guidée par corpus et fondée sur corpus), nous nous intéressons aux écarts de réalisations avec la « norme », selon l’origine des locuteurs. Des analyses acoustiques et perceptives conduisent à la création d’outils intégrés de formalisation, de représentation, de remédiation et d’évaluation selon la langue d’origine de chaque apprenant. The PhoDiFLE corpus has been developed to enable researchers, especially those concerned with remedial phonetics, to compare French native speakers and foreign learners utterances in French. This article examines the issues that had to be dealt with while creating this corpus. Our approach combines corpus-driven and corpus-based studies and focuses on the phonetic variants across native and non-native speakers. Acoustic and perceptual analyses allow to compare their productions. This corpus will finally result in the making of tools aiming to formalise, represent, correct and assess the variants observed in learners of French as a Second Language.

p = 0.12) than that in the cortex but did not reach statistical difference. Renal ketogenesis increases during hepatic inflow occlusion with AR administration. It is likely that the enhancement of ketogenesis takes place predominantly in the medulla of the kidney.RID="" ID="" Correspondence to: T. Nakatani, M.D., Department of Emergency and Critical Care Medicine, Kansai Medical University, 10-15 Fumizonocho, Moriguchi, Osaka 570-8507, Japan

Background: Intra-abdominal hypertension is known to decrease hepatic blood flow, but its effect on hepatic energy level has not described. Methods: Fifty-three rabbits were mechanically ventilated and divided into five groups. Intra-abdominal hypertension was induced by saline infusion and maintained for 30 minutes. Hepatic sinusoidal functional blood flow was evaluated by means of indocyanine green disappearance rate (ICG-K), hepatic mitochondrial redox status was evaluated by arterial ketone body ratio, and tissue energy level was evaluated by energy charge (EC). Results: At an intra-abdominal pressure of 20 mm Hg, ICG-K was significantly decreased, with no decrease in EC. At 30 mm Hg, hypoxemia developed and the ICG-K decreased further, with significant decreases observed in arterial ketone body ratio and EC. The latter were not increased by administration of oxygen. Conclusion: At an intra-abdominal pressure of 20 mm Hg, a slight decrease in sinusoidal flow did not affect hepatic energy level. At 30 mm Hg, a reduced hepatic mitochondrial redox status and a decreased energy level were attributed to a decrease in sinusoidal flow in this animal model.

We investigated the effects of pretreatment with a potent platelet-activating factor (PAF) receptor antagonist (E5880) on the changes in hepatic and systemic metabolism induced by transient hepatic ischemia and reperfusion. Sixty-five rabbits were assigned to four groups that either did or did not undergo a period of hepatic ischemia and reperfusion with or without pretreatment. E5880 was administered intraportally 1 minute prior to inflow occlusion. Twenty minutes of warm ischemia was followed by 30 minutes of reperfusion. Blood gas analyses and measurements of levels of arterial pyruvate, lactate, and ketone bodies, arterial and portal ammonia and endotoxin, and intrahepatic adenine nucleotide, pyruvate, and lactate were performed. Results were analyzed by either ANOVA or chi-square analysis. Hepatic tissue ATP and energy charge levels were significantly increased and the AMP level was significantly decreased after 30 minutes of reperfusion in the pretreatment group compared to those without pretreatment. At the same time, parameters reflecting hepatic mitochondrial function, such as the arterial ketone body ratio and arterial ammonia level, improved, although they were not statistically significant. No difference was observed for parameters reflecting systemic changes, such as arterial blood gas values and pyruvate and lactate levels. PAF is thought to mediate metabolic changes after hepatic ischemia and reperfusion. PAF released in the liver may exert local effects, which appear to be attenuated by pretreatment with E5880. Systemic metabolic changes seen after hepatic ischemia and reperfusion may be mediated by factors other than PAF.

The blood levels of ketone bodies, which are synthesized principally in the liver, were maintained even during hepatic inflow occlusion if Ringer's acetate solution (AR) was administered, resulting in an improvement of hepatic energy level in the reperfusion phase, as reported in our previous experimental study. The current study was designed to prove that the kidneys are the organs that contribute to synthesize ketone bodies during hepatic inflow occlusion if AR is administered.The arterial, central venous, renal venous, and renal tissue ketone body concentrations were determined in rabbits administered AR or Ringer's lactate solution (LR) at 20 minutes of hepatic ischemia and at 30 minutes of reperfusion. The concentrations were also compared in rabbits under AR infusion with or without hepatic ischemia for 20 minutes. Statistical analyses were performed by means of ANOVA:With AR the renal venous ketone body concentration not only was higher than that with LR (p0.001) but also was higher than the arterial concentration (p = 0.05). The renal tissue ketone body concentration was higher than in those with LR (p0.001) and also than in those without occlusion (p0.001).Ketogenesis is enhanced in the kidney and may compensate for hepatic loss of ketogenic function during hepatic inflow occlusion under AR administration.

Objective : We investigated the relation between in vivo hepatocyte excretion of bile and bilirubin and hepatic energy status in rabbit models of hemorrhagic shock and hypoxemia. Design : Randomized animal study. Materials and Methods : After creation of a total biliary fistula, hemorrhagic shock with mean pressure of 50 mm Hg (10 rabbits) or hypoxemia with Pao 2 at 35 mm Hg (8 rabbits) was induced for 60 minutes. We determined bile flow, excretion of bilirubin and total bile acids, the plasma level of bilirubin, and arterial ketone body ratio, which reflects hepatic mitochondrial function. Measurements and Main Results : Both the hemorrhagic shock and the hypoxemic models showed decreases in bile flow and excretion of bilirubin and total bile acids as well as increase in the plasma level of bilirubin in association with decreases in the hepatic energy charge and the arterial ketone body ratio. Conclusions : Bile flow and the excretion of bilirubin were correlated with the hepatic energy status.

This study aimed to clarify the difference in the effects of sodium acetate and sodium lactate administration on hepatic energy metabolism during hepatic warm ischemia and reperfusion. In the first experiment, Ringer's acetate (AR) or Ringer's lactate (LR) solutions were administered intravenously during 20 min of hepatic inflow occlusion in rabbits. Blood gas analyses and measurements of blood pressure, pyruvate, lactate, and ketone body concentrations in arterial blood were performed until 30 min of reperfusion. Hepatic tissue adenine nucleotide concentrations were determined at the end of the experiment. With AR administration, the plasma pyruvate level and the ratio of pyruvate to lactate were significantly elevated during reperfusion. Hepatic energy charge at 30 min of reperfusion improved significantly (P < 0.001) with AR compared with LR administration. Plasma ketone body concentrations decreased markedly with LR administration, but were maintained with AR administration. In the second experiment, intravenous administration of AR or LR, hepatic ischemia, and reperfusion were similarly performed in rabbits. Ketone body concentrations were determined in arterial and vena cava blood at 0 min, 20 min of inflow occlusion, and 30 min of reperfusion. With AR administration, a large arteriovenous difference in ketone body concentrations was observed, indicating utilization in the peripheral tissues. With LR administration, no arteriovenous difference was observed. Availability of energy substrate in peripheral tissues by administration of AR is thought to decrease the metabolic load to the liver and to improve hepatic energy status during reperfusion.