Prevention of symptomatic pulmonary embolism for gynecologic malignancies with preoperative asymptomatic venous thromboembolism: GOTIC-VTE trial.

OBJECTIVE: In Japan, perioperative prophylaxis of pulmonary embolism (PE) in gynecologic cancer patients with preoperative asymptomatic venous thromboembolism (VTE) has not been well established yet. The GOTIC-VTE trial was a prospective, multi-center, single-arm clinical trial to investigate the prevention of postoperative symptomatic PE onset by seamless anticoagulant therapy from the preoperative period to 4 weeks after surgery instead of using intermittent pneumatic compression. METHODS: Anticoagulant therapy was started immediately after asymptomatic VTE diagnosis and stopped preoperatively according to the rules of each institution. Unfractionated heparin administration was resumed within 12 hours postoperatively, and this was followed by the switch to low-molecular-weight heparin and subsequently, edoxaban; this cycle was continued for 28 days. Primary outcome was the occurrence of symptomatic PE in 28 days postoperatively. Secondary outcomes were the incidence of VTE-related events in 28 days and 6 months postoperatively and protocol-related adverse events. RESULTS: Between February 2018 and September 2020, 99 patients were enrolled; of these, 82 patients were assessed as the full analysis set, including 58 for ovarian cancer, fallopian tube, or peritoneal cancer; 21 for endometrial cancer; and 3 for cervical cancer. No symptomatic PE was observed within 28 days postoperatively; two patients had bleeding events (major bleeding and clinically relevant nonmajor bleeding) and three had grade 3 adverse events (increased alanine transaminase, aspartate aminotransferase, or gamma-glutamyl transferase). CONCLUSION: The multifaceted perioperative management for gynecologic malignancies with asymptomatic VTE effectively prevented postoperative symptomatic PE. TRIAL REGISTRATION: JRCT Identifier: jRCTs031180124.

Anti-nanodisc antibodies specifically capture nanodiscs and facilitate molecular interaction kinetics studies for membrane protein.

Nanodisc technology has dramatically advanced the analysis of molecular interactions for membrane proteins. A nanodisc is designed as a vehicle for membrane proteins that provide a native-like phospholipid environment and better thermostability in a detergent-free buffer. This enables the determination of the thermodynamic and kinetic parameters of small molecule binding by surface plasmon resonance. In this study, we generated a nanodisc specific anti-MSP (membrane scaffold protein) monoclonal antibody biND5 for molecular interaction analysis of nanodiscs. The antibody, biND5 bound to various types of nanodiscs with sub-nanomolar to nanomolar affinity. Epitope mapping analysis revealed specific recognition of 8 amino acid residues in the exposed helix-4 structure of MSP. Further, we performed kinetics binding analysis between adenosine A2a receptor reconstituted nanodiscs and small molecule antagonist ZM241385 using biND5 immobilized sensor chips. These results show that biND5 facilitates the molecular interaction kinetics analysis of membrane proteins substituted in nanodiscs.

Utility of vaginal vault cytology in the local recurrence of cervical cancer.

BACKGROUND: In Japan, 8000 women were newly diagnosed with cervical cancer in 2018. The healthcare insurance policy in Japan allows physicians to utilize vaginal volt cytology tests and serum biomarker measurement at every visit and imaging analysis at an adequate interval with screening for recurrence after initial treatment. However, the major surveillance guidelines published in the United States and European countries recommend focusing on pelvic examinations and symptom reviews to avoid unnecessary tests. This study aimed to reassess the benefits of standard surveillance methods adopted in this study by retrospective analysis. METHODS: From January 2009 to December 2015, the medical records of patients with recurrence who were initially diagnosed with International Federation of Gynecology and Obstetrics stage I-III cervical cancer were collected for this study. Clinicopathological data were statistically analyzed to identify significant factors. In the first 2 years, the patients underwent regular surveillance, including pelvic examination, serum tumor marker tests, vaginal vault cytology every 1-3 months, and imaging analysis at 6- to 12-month intervals. In the following 2 years, the patients received a regular check with the same methods every 4 months and an annual imaging analysis. Afterward, the patients had regular screening every 6 to 12 months. RESULTS: In the study period, 84 of the 981 patients experienced recurrence, and 88.1% had an asymptomatic recurrence. The disease-free interval was not related to the recurrence site. In univariate analysis, primary treatment, recurrence site, and diagnostic method were significant factors for survival outcomes. In contrast, multivariate analysis indicated that only primary treatment was a significant factor. In patients with local recurrence, multivariate analysis demonstrated that radiation as salvage therapy was an independent predictive factor for overall survival after recurrence. CONCLUSIONS: In this retrospective study, routine imaging analysis and serum biomarker measurement did not contribute to patient prognosis after recurrence. In contrast, vaginal vault cytology can improve survival after recurrence in some patients. Tailored surveillance methods based on individual disease conditions and treatment modalities can improve post-recurrent survival outcomes.

Reassessment of intensive surveillance practices adopted for endometrial cancer survivors.

BACKGROUND: In Japan, 17,000 women are newly diagnosed with endometrial cancer in 2018. The healthcare insurance policy in Japan provides more intensive patient surveillance compared with the United States and European countries. The aim of this study was to retrospectively analyze data, including surveillance methods, recurrence sites, salvage therapy, and survival period after recurrence, to consider the benefits of surveillance for patients with endometrial cancer. METHODS: Between January 2009 and December 2015, the medical records of patients who were initially diagnosed with the International Federation of Gynecology and Obstetrics stage I-IV endometrial cancer and treated were enrolled in this retrospective study. Only patients with stage IV cancer with peritoneal dissemination were included. Within the first 2 years, the included patients underwent tumor marker tests, Papanicolaou smear test every 1-3-months, and imaging analysis at 6-12- month intervals. Until 4 years, the patients underwent regular surveys every 4 months and imaging analysis annually. Subsequently, the patients received regular surveys every 6 -to 12-months. RESULTS: Among 847 patients, 88 experienced recurrence, and their clinicopathological data were statistically analyzed. The recurrence site was not associated with the initial treatment method or histology. Among the patients with recurrence, 75% were asymptomatic. Univariate analysis demonstrated that time to recurrence and local recurrence were significant factors for survival outcomes, whereas multivariate analysis indicated that only local recurrence was a significant factor. In patients with distant metastasis, neither symptomatic nor asymptomatic recurrence showed a significant difference in survival. CONCLUSIONS: In this retrospective study, an intensive surveillance protocol did not benefit patients with endometrial cancer. Thus, we hypothesize that the characterization of tumors by emerging technologies that can precisely predict the nature of the tumor will help tailor individualized and efficient surveillance programs. In addition, the ideal salvage therapy needs to be developed to benefit patients after recurrence.

Nadir CA-125 serum levels during neoadjuvant chemotherapy and no residual tumor at interval debulking surgery predict prognosis in advanced stage ovarian cancer.

BACKGROUND: Recent phase III randomized trials have suggested that neoadjuvant chemotherapy followed by interval debulking surgery (NACT-IDS) is a treatment option for patients with advanced epithelial ovarian cancer. This study aimed to use CA-125 and computed tomography (CT) scanning to generate a simple and clinically applicable model of predicting complete cytoreduction by interval debulking surgery (IDS) and the overall survival in patients who receive taxane/platinum-based chemotherapy as neoadjuvant chemotherapy (NACT). METHODS: Patients with stage IIIc or IV epithelial ovarian cancer who underwent taxane/platinum-based NACT followed by IDS in Gunma Prefectural Cancer Center, Takasaki General Medical Center, and Gunma University from April 2009 to March 2015 were included. Patients underwent a CT scan to confirm confirm tumors unresectable by standard surgery before NACT. CA-125 levels were measured pre-NACT, after each cycle of NACT, and before IDS. CT was also performed before IDS to evaluate tumor metastasis. Data were collected retrospectively and analyzed to determine the predictive factors of complete resection and overall survival. RESULTS: Among 63 patients who received NACT-IDS, 43 and 20 patients had stages IIIc and IV epithelial ovarian cancer at diagnosis, respectively. CT predictors of residual tumors after IDS such as extra-ovarian implants (P = 0.009) and omental cakes (P = 0.038) were not present. Univariate analysis revealed that the independent factors for overall survival were no residual tumor by IDS (P = 0.0016) and CA125 ≤ 20 U/ml before IDS (P = 0.0011). CONCLUSIONS: Although this study had a small sample size, NACT-IDS used to completely remove macroscopic disease which significantly improved the prognosis of patients with preoperative CA-125 ≤ 20 U/ml. Results from this study provide useful information for future studies on the management of patients with advanced epithelial ovarian cancer.

High Expression of Ubiquitin C-terminal Hydrolase L1 Is Associated With Poor Prognosis in Endometrial Cancer Patients.

OBJECTIVE: The ubiquitin C-terminal hydrolase L1 (UCHL1) plays a key role in tumor invasion and metastasis. Ubiquitin C-terminal hydrolase L1 is overexpressed in various cancers and reported to be correlated with a poor prognosis. The objective of this study was to determine the prognostic significance of UCHL1 in endometrial cancer. METHODS: The expression of UCHL1 in endometrial cancer was assessed using quantitative reverse transcription polymerase chain reaction and immunohistochemistry in 56 and 215 resected tumor specimens, respectively. RESULTS: The 4-year survival rates of the high UCHL1 messenger RNA expression group and high UCHL1 protein expression group were 78% and 71%, respectively, compared with 96% and 95% for the low UCHL1 messenger RNA expression group and low UCHL1 protein expression group, respectively. Kaplan-Meier and log-rank tests indicated a significant correlation between expression of UCHL1 and disease-free survival and overall survival. Moreover, multivariate stepwise Cox proportional hazard regression model analysis showed that UCHL1 was a significant independent marker for predicting a poor disease-free survival and overall survival. In 43 patients with metastatic lesions, immunohistochemical analysis of metastatic lesions revealed that the recurrence rate and mortality rate were 62% and 41%, respectively, in 29 UCHL1-positive patients and 36% and 29%, respectively, in 14 UCHL1-negative patients. CONCLUSIONS: The results of this study suggest that high UCHL1 expression is a strong marker of poor prognosis of endometrial cancer. Furthermore, we suggest that UCHL1 may be involved in the development of distant metastasis in endometrial cancer.

Effect of the molecular targeted drug, erlotinib, against endometrial cancer expressing high levels of epidermal growth factor receptor.

BACKGROUND: The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, erlotinib, has been clinically applied for the treatment of a variety of tumors with EGFR overexpression. A phase II clinical study of erlotinib (NCIC IND-148) for recurrent or metastatic endometrial carcinoma (EC) resulted in an unfavorable result. However, in that study, the expression levels of EGFR were not accurately analyzed. Thus, the aim of this study was to re-examine the efficacy of erlotinib in EC cells by utilizing in vitro and in vivo models. METHODS: Tissue samples obtained from patients histologically diagnosed with EC of the uterine corpus were subjected to immunohistochemistry and RT-PCR to determine the protein and mRNA expression levels of EGFR. Western blot and WST-1 assays of EGFR siRNA-transfected HEC-1A, KLE, and Ishikawa cells were used to evaluate the efficacy of erlotinib in tumor cell lines expressing different EGFR levels. Furthermore, HEC-1A and Ishikawa cells were implanted into athymic mice treated with either erlotinib or trastuzumab. RESULTS: At our institution, 20.9% of endometrial cancer patients with low grade endometrioid histology have been diagnosed as stage III and IV. Immunohistochemical analysis and RT-PCR revealed the presence of significant EGFR and EGFR mRNA expression in low-grade endometrioid carcinoma in comparison with high-grade endometrioid carcinoma. In vitro study, WST-1 assay and Western blot analysis revealed that EGFR expression levels were correlated with tumor cell viability. Erlotinib reduced the proliferation of HEC-1A expressing high levels of EGFR, while trastuzumab showed similar effect in Ishikawa cells dominantly expressing human epidermal growth factor receptor type2 (HER2). In vivo erlotinib decreased tumor growth in mice xenografted with HEC-1A cells, whereas this tumor-growth inhibition was not observed in trastuzumab-treated mice xenografted with Ishikawa cell. CONCLUSIONS: EGF contributed to tumor proliferation in EC cell lines along with EGFR expression in vitro. Erlotinib also demonstrated anti-tumor effects in xenograft mice models. Our results suggest that erlotinib continues to have clinical usefulness in specific cases, after taking into consideration the EGFR expression levels.

The ERK1/2 pathway regulates testosterone synthesis by coordinately regulating the expression of steroidogenic genes in Leydig cells.

Adult mice with a Leydig cell specific deletion of MAPK kinase (MEK) 1 and 2 (Mek1(f)(/)(f);Mek2(-/-);Cre(+)) mice display Leydig cell hypoplasia and hypergonadotropic hypogonadism. We used radioimmunoassays and quantitative PCR to evaluate the function and expression of the Leydig cell genes involved in the conversion of cholesterol to testosterone (Star, Cyp11a1, Hsd3b6, Cyp17a1 and Hsd17b3), androgen metabolism (Srda1 and Dhrs9), and four transcription factors (Creb1, Nr5a1, Nr4a1 and Nr0b1) that regulate the expression of steroidogenic genes. We show that Star, Hsd3b6, Cyp17a1 and Hsd17b3 are downregulated in Ledyig cells of adult Mek1(f)(/)(f);Mek2(-/-);Cre(+) mice whereas Srda1 and Dhrs9 are upregulated and Creb1, Nr5a1, Nr4a1 and Nr0b1 are unchanged or upregulated. Functionally, all the downregulated genes but none of the upregulated genes contribute to the decrease in testosterone synthesis in Leydig cells of adult Mek1(f)(/)(f);Mek2(-/-);Cre(+) mice because they produce low testosterone and dihydrotestosterone when stimulated with hCG or when incubated with testosterone precursors such as progesterone or androstenedione.

The Leydig cell MEK/ERK pathway is critical for maintaining a functional population of adult Leydig cells and for fertility.

MAPK kinase (MEK)1 and MEK2 were deleted from Leydig cells by crossing Mek1(f/f);Mek2(-/-) and Cyp17iCre mice. Primary cultures of Leydig cell from mice of the appropriate genotype (Mek1(f/f);Mek2(-/-);iCre(+)) show decreased, but still detectable, MEK1 expression and decreased or absent ERK1/2 phosphorylation when stimulated with epidermal growth factor, Kit ligand, cAMP, or human choriogonadotropin (hCG). The body or testicular weights of Mek1(f/f);Mek2(-/-);iCre(+) mice are not significantly affected, but the testis have fewer Leydig cells. The Leydig cell hypoplasia is paralleled by decreased testicular expression of several Leydig cell markers, such as the lutropin receptor, steroidogenic acute regulatory protein, cholesterol side chain cleavage enzyme, 17α-hydroxylase, and estrogen sulfotransferase. The expression of Sertoli or germ cell markers, as well as the shape, size, and cellular composition of the seminiferous tubules, are not affected. cAMP accumulation in response to hCG stimulation in primary cultures of Leydig cells from Mek1(f/f);Mek2(-/-);iCre(+) mice is normal, but basal testosterone and testosterone syntheses provoked by addition of hCG or a cAMP analog, or by addition of substrates such as 22-hydroxycholesterol or pregnenolone, are barely detectable. The Mek1(f/f);Mek2(-/-);iCre(+) males show decreased intratesticular testosterone and display several signs of hypoandrogenemia, such as elevated serum LH, decreased expression of two renal androgen-responsive genes, and decreased seminal vesicle weight. Also, in spite of normal sperm number and motility, the Mek1(f/f);Mek2(-/-);iCre(+) mice show reduced fertility. These studies show that deletion of MEK1/2 in Leydig cells results in Leydig cell hypoplasia, hypoandrogenemia, and reduced fertility.

Lymphangioleiomyomatosis suspected to be a gynecologic disease.

Lymphangioleiomyomatosis (LAM) is a rare, systemic disorder that predominantly affects women. Although patients with LAM mostly present with pulmonary symptoms, some patients may present initially with extrapulmonary symptoms. We present a case of a 30-year-old Japanese female with abdominal pain during menstrual periods was suspected of having ovarian cancer due to exaggerated ascites observed at a local clinic. Subsequently, she was transferred to our hospital for further investigations, and was diagnosed with LAM. Three years after diagnosis, she had a girl by cesarean section to avoid the progression of pulmonary LAM by vaginal delivery. The patient is undergoing follow-up treatment with the administration of gonadotropin-releasing hormone-analog. Though LAM is rare, gynecologists should know about it because it may occur with gynecological symptoms in young women.

Effect of estrogen on the expression of luteinizing hormone-human chorionic gonadotropin receptor messenger ribonucleic acid in cultured rat granulosa cells.

Estrogen has been considered to enhance FSH actions in the ovary, including the induction of the LH receptor (LHR). In this study, we elucidated the mechanism underlying the effect of estrogen on the induction of LHR by FSH in rat granulosa cells. Estradiol clearly enhanced the FSH-induced LHR mRNA increase in a time- and dose-dependent manner, with a maximum increase of approximately 3.5-fold at 72 h, compared with the level of LHR mRNA solely induced by FSH. We then investigated whether the effect of estrogen on LHR mRNA was due to increased transcription and/or altered mRNA stability. A luciferase assay with the plasmid containing the LHR 5'-flanking region did not show that estradiol increased the promoter activity induced by FSH. In contrast, the decay curves for LHR mRNA showed a significant increase in half-life with FSH and estradiol, suggesting that the increased stability of LHR mRNA is at least responsible for the regulation of LHR mRNA by estrogen. Recently mevalonate kinase (Mvk) was identified as a trans-factor that binds to LHR mRNA and alters LHR mRNA stability in the ovary. We found that estradiol, with FSH, decreased Mvk mRNA levels in rat granulosa cell culture, resulting in up-regulation of LHR mRNA that was inversely correlated to Mvk mRNA expression. Furthermore, the augmentation of FSH-induced LHR expression in the presence of estrogen was erased with the overexpression of Mvk by transient transfection. Taken together, these data indicate that LHR mRNA is up-regulated due to increased stability when estrogen negatively controls Mvk.

Regulation of human luteinizing hormone receptor in the ovary.

The luteinizing hormone receptor (LHR) is essential for elevated levels of progesterone to maintain pregnancy during the first trimester; the maintenance of the expression of LHR is a key factor controlling the duration of luteal function. Therefore, as the expression of LHR is most likely to be regulated by the stability of the receptor mRNA at the luteal phase of the human menstrual cycle, we focused on studies examining the stability of mRNA rather than the production of mRNA. In addition, LHR (exon 9), one of the splice variants of human LHR (hLHR), was cloned in the corpus luteum of a patient with a regular menstrual cycle. The results of Western blots using Percoll gradient fractionation indicated that hLHR formed complexes with hLHR (exon 9), which are transferred to the lysosome, where they are eventually degraded, instead of being translocated from the endoplasmic reticulum to the transducing organelle. These results showed that hLHR (exon 9) caused a reduction in the expression of functional receptor number and affected the signaling condition of wild-type hLHR. As the luteal phase progressed hLHR (exon 9) increased relative to hLHR, demonstrating that hLHR (exon 9) was expressed more than hLHR in the late luteal phase. This work reveals the essential function of the regulatory and structural elements involved in human LH receptor splicing, and that hLHR (exon 9) can negatively control the function of wild-type receptors. Moreover, this finding presented a novel mechanism of regulation of LHR in the human corpus luteum. (Reprod Med Biol 2008; 7: 11-16).

The effect of splice variant of the human luteinizing hormone (LH) receptor on the expression of gonadotropin receptor.

A splice variant of human lutropin/choriogonadotropin-receptor [hLHR (exon 9)] that lacks exon 9 was previously cloned in the corpus luteum of a woman with a regular menstrual-cycle. Supported by detergent soluble binding assay and receptor biotinylation experiment, the receptor binding assay shows hLHR (exon 9) is neither expressed at the cell surface nor have the capability of binding to hCG. Interactions between hLHR (exon 9) with the immature bands of gonadotropin receptors not with the mature bands were seen. This phenomenon is specific among gonadotropin receptors since human thyrotropin-receptor (hTSHR) failed to be coimmunoprecipitated. Furthermore, this receptor complex attenuated the receptor protein level within the cells. To elucidate the mechanism underlying the decrease in receptor protein by this receptor complex, we performed a Percoll-fractionation experiment, which indicated the receptor complex drove hLHR to the lysosome instead of the plasma-membrane. Moreover, the expression of hLHR (exon 9) mRNA was seen at all phases of the menstrual cycle and relatively increased as the luteal phase progressed. These results reveal a novel mechanism of regulation for gonadotropin receptor expression.

Effect of follicle-stimulating hormone and estrogen on the expression of betaglycan messenger ribonucleic acid levels in cultured rat granulosa cells.

Betaglycan (TGFbeta type III receptor) was recently identified as a coreceptor to enhance the binding of inhibin A to activin type II receptor. This inhibin/betaglycan/activin type II receptor complex prevents activins from binding to their own receptors. The present study was undertaken to identify the expression and the regulation of the betaglycan gene in cultured rat granulosa cells. Northern blot analysis indicated betaglycan mRNA transcript of approximately 6.4 kbp. The treatment of the cells with FSH increased the betaglycan mRNA level, and a concurrent treatment with estradiol brought a significant increase in betaglycan mRNA. The protein kinase A activator, 8-bromoadenosine-cAMP, also increased the expression of its mRNA. Furthermore, betaglycan mRNA was induced additively by estradiol, which was blocked by estrogen receptor antagonists [ICI 182780, (R, R)-cis-diethyltetrahydro-2,8-chrysenediol]. In the luciferase assay, FSH altered the promoter activity of betaglycan. Moreover, when FSH plus estradiol was added to the granulosa cells, a significant increase in the half-life of betaglycan mRNA transcript was seen. In summary, FSH and estradiol increased betaglycan mRNA expression, most possibly through the protein kinase A pathway and the estrogen receptor-beta. The increase of betaglycan mRNA was due to an increase in transcription and altered mRNA stability. In ovarian regulatory function, the expression of betaglycan may involve the functional antagonism of inhibin A in activin signal transduction.

Association of human follitropin (FSH) receptor with splicing variant of human lutropin/choriogonadotropin receptor negatively controls the expression of human FSH receptor.

A splice variant of human lutropin (LH)/choriogonadotropin (CG)-receptor [hLHR(exon 9)] that lacks exon 9 was previously cloned in the corpus luteum of a woman with a normal menstrual cycle. Supported by a detergent-soluble binding assay and a receptor biotinylation experiment, the receptor binding assay shows hLHR(exon 9) is neither expressed at the cell surface nor has the capability of binding to hCG. In addition, hLHR(exon 9) was confirmed in the endoplasmic reticulum (ER) by endoglycosidase H treatment. A coimmunoprecipitation experiment clearly showed that hLHR(exon 9) and constitutively inactivate mutant-LHRs, which stay in the ER, form an association with the human follitropin (FSH)-receptor (hFSHR). This suggests that in the presence of mutant-LHR, hFSHR, which is trapped in the ER and associated with hLHR(exon 9), is unable to come up to the plasma membrane. This phenomenon is specific among gonadotropin receptors because human TSH receptor failed to be coimmunoprecipitated. Furthermore, this receptor complex attenuated the hFSHR receptor protein level within the cells, which impaired cAMP production. To elucidate the mechanism underlying the decrease in hFSHR protein by this receptor complex, we performed a Percoll fractionation experiment, which indicated that the receptor complex drove hFSHR to the lysosome instead of the plasma membrane. These results reveal a novel mechanism of FSHR expression regulation.

A splice variant of the human luteinizing hormone (LH) receptor modulates the expression of wild-type human LH receptor.

We previously reported a splice variant form of human LH receptor [hLHR(exon 9)] that lacks exon 9, coding the N-terminal extracellular region close to the first transmembrane domain. Several recent studies suggest that G protein-coupled receptors are able to form dimerization or oligomerization of the receptor, suggesting an intermolecular interaction between hLHR(exon 9) and the wild-type LH receptor (hLHR). The aim of this study, using coimmunoprecipitation, is to examine whether hLHR forms an association with hLHR(exon 9). An interaction between hLHR(exon 9) with the immature band (68 kDa) of hLHR and not with the mature band (85 kDa) was seen. When hLHR and hLHR(exon 9) were coexpressed, the density of hLHR expression was significantly reduced, compared with hLHR expressed alone. The human chorionic gonadotropin-stimulated cAMP accumulation in the cells expressing hLHR(exon 9) was also impaired, compared with the cells expressing hLHR. In this study, we demonstrated that hLHR is capable of forming receptor complexes. Our findings may expand the possibility of a splice variant of hLHR specifically modulating the functional property of the wild-type hLHR.