The role of non-genomic actions of progesterone and its membrane receptor agonist in ovarian cancer cell death.

BACKGROUND: Progesterone therapy is a relatively inexpensive treatment option for endometrial and breast cancers, with few side effects. Two signaling pathways usually mediate the physiological effects of progesterone, namely genomic and non-genomic actions. Genomic action occurs slowly via the nuclear progesterone receptor (PR), whereas the membrane progesterone receptor (mPR) induces rapid non-genomic action. AIMS: We investigated the effects of progesterone and various PR agonists on ovarian cancer cells. METHODS AND RESULTS: PR expression of six serous ovarian cancer cell lines was examined by western blotting, and mPR expression was examined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). PR-negative and mPR-positive ovarian cancer cells were exposed to progesterone and seven types of PR agonists (medroxyprogesterone acetate [MPA], dehydroepiandrosterone, dienogest, levonorgestrel, drospirenone, pregnenolone, and allopregnanolone) at 10-400 µM, and viable cell counts after exposure for 30 min were measured using the water-soluble tetrazolium (WST-1) assay. Ovarian cancer cell lines were exposed to 100 µM progesterone, and the expression of BAX, a pro-apoptotic protein, after 1-5 min was examined by western blotting. Western blotting detected no PR expression in the six serous ovarian cancer cell lines. In contrast, RT-qPCR detected mPR expression in all six serous ovarian cancer cell lines. Progesterone and MPA-induced cell death in all tested ovarian cancer cell lines in a concentration-dependent manner, whereas no effect was observed for other PR agonists. Western blotting revealed that pro-apoptotic protein BAX expression occurred 1 min after exposure to progesterone, suggesting that the cytocidal effects are mediated by rapid non-genomic action. CONCLUSION: Progesterone and MPA exhibited a rapid cytocidal effect on PR-negative ovarian cancer cells through non-genomic action. Progesterone and MPA could be novel adjuvant therapies for ovarian cancer.

Ovarian vein thrombosis after bilateral adnexectomy in a symptomatic patient with concomitant pulmonary embolism: A case report.

OBJECTIVE: Ovarian vein thrombosis (OVT) after adnexectomy is usually asymptomatic, and pulmonary embolism (PE) has not been reported following this type of OVT. We present the case of a patient with symptomatic OVT after bilateral adnexectomy who experienced PE. CASE REPORT: A 52-year-old woman underwent total laparoscopic hysterectomy and bilateral adnexectomy for early stage endometrial cancer. On the 12th postoperative day, she presented with a fever of 38.7 °C. Computed tomography (CT) revealed bilateral OVT. Anticoagulant and antibacterial therapy was initiated; after five days, the fever subsided. On the 19th postoperative day, CT revealed a decrement in OVT; however, PE was observed. By the 60th postoperative day, PE disappeared. No deep vein thromboses were detected at any time. CONCLUSION: This case highlights that OVT, even after adnexectomy, can cause symptoms and PE can occur after this type of OVT. Anticoagulation therapy may be considered in such cases.

A patient with a mucinous borderline ovarian tumor after fertility-sparing surgery in whom puncture fluid cytology on oocyte retrieval led to a diagnosis of recurrence.

The patient was an unmarried nulliparous 21-year-old female who was referred to our hospital with an abdominal mass. Bilateral ovarian tumors with a solid component were detected and both were suspected to be ovarian cancer. Since the patient strongly wished to preserve fertility, we performed left salpingo-oophorectomy, right cystectomy, and omentectomy. A postoperative histopathological examination revealed that the bilateral ovarian tumors were mucinous borderline tumors. The patient selected oocyte cryopreservation. Oocyte retrieval from the right ovary was performed 2 years after surgery, and six oocytes were obtained. The puncture of a small cyst revealed mucinous fluid, not normal follicular fluid; therefore, puncture fluid cytology was performed. A recurrent mucinous borderline ovarian tumor was suspected. Right salpingo-oophorectomy was performed and a recurrent mucinous borderline ovarian tumor was diagnosed. Puncture fluid properties need to be considered when there is an opportunity for oocyte retrieval after fertility-sparing surgery.

Durable response after the discontinuation of pembrolizumab treatment due to an adverse event in a patient with advanced endometrial cancer: A case report.

The persistence of antitumor effects has been reported after the completion of treatment with immune checkpoint inhibitors (ICIs) for various types of carcinoma, such as malignant melanoma, exhibiting a durable response. A durable response has also been noted after the discontinuation of treatment at an early stage due to adverse events, including in renal pelvic cancer, pancreatic cancer and intrahepatic cholangiocarcinoma; however, to the best of our knowledge, a similar case report has not yet been published in the malignant gynecological tumor field. The present study described a patient with refractory advanced endometrial cancer in whom the administration of pembrolizumab was discontinued after the completion of the 7th course due to renal dysfunction; however, persistent tumor-reducing effects and decreases in the levels of tumor markers were noted for more than 18 months after the cessation of treatment. Pembrolizumab may be continuously administered to some patients for a long period, whereas a durable response is achieved by others even after its discontinuation at an early stage; therefore, difficulties are associated with selecting an appropriate duration of administration. Further studies are required to search for biomarkers that facilitate high-accuracy effect predictions, and to establish an optimal administration period in consideration of specific adverse reactions to ICIs and cost-effectiveness.

Response to and toxicity of weekly paclitaxel and carboplatin in patients with stage IIIC-IV ovarian cancer and poor general condition.

It has remained elusive whether standard chemotherapy regimens are safe for patients with ovarian cancer and poor general condition. The purpose of the present study was to assess the response to and toxicity of weekly paclitaxel and carboplatin (W-PC) in patients with ovarian cancer and poor general condition. The subjects were patients with ovarian cancer who received W-PC at Jichi Medical University Hospital (Shimotsuke, Japan) between January 2008 and December 2016. Patients who were ≥80 years old and/or had a performance status ≥3 and/or severe complications/underlying diseases were selected. Patients received paclitaxel (60 mg/m2) and carboplatin (area under the curve 2 mg/ml/min) on days 1, 8, and 15 of a 28-day cycle. Their medical records were retrospectively reviewed. A total of 31 patients were included in the study. Grade 3/4 neutropenia, anemia and thrombocytopenia developed in 18 (58%), 5 (16%) and 1 (3%) patients, respectively. Furthermore, three (10%) patients had a complete response (CR), 12 (39%) had a partial response (PR), 5 (16%) had stable disease and 11 (35%) had progressive disease. The overall response rate was 48% (15/31) and the disease control rate was 65% (20/31). The 5-year progression-free survival was 15% and the 5-year overall survival was 15%. A total of 9 patients survived for >40 months, one of whom survived without recurrence for 122 months. Performance status <3, a tumor response of CR or PR and >5 chemotherapy cycles were indicators of favorable prognosis. Only >5 chemotherapy cycles (vs. ≤5; P=0.002) was an independent good prognostic factor according to multivariate analysis. In conclusion, W-PC was tolerable and slightly effective in patients with ovarian cancer and poor general condition. W-PC may be one option for patients who are unable to receive standard chemotherapy regimens.

Prognostic significance of the number of removed lymph nodes according to FIGO stage in ovarian clear cell carcinoma.

A previous study by our group reported that removing a larger number of lymph nodes in patients with stage I ovarian clear cell carcinoma (OCCC) improved progression-free survival (PFS). The present study investigated whether clinical conditions, particularly the number of removed lymph nodes, are independent predictors of progression for stage II or higher OCCC and whether the significance of the number of removed lymph nodes differs according to FIGO stage for OCCC. A total of 113 patients with OCCC who had undergone surgery between January 1993 and December 2015 were retrospectively enrolled and the clinicopathological data were obtained from their medical records. Among patients with stage II or higher OCCC, PFS of those with no residual tumor or no lymph node metastasis was significantly better than that of those with residual tumor (P=0.023) or lymph node metastasis (P=0.035). Multivariate analysis revealed that no residual tumor was the only independent predictor for improved PFS of patients with stage II or higher. Regarding the number of removed lymph nodes, it did not significantly affect the PFS of patients with stage II or higher OCCC, whereas it improved the PFS of those with stage I, being an independent predictor of progression of stage I OCCC. In summary, although the number of removed lymph nodes was an independent predictor of progression for stage I OCCC, it was not for stage II or higher OCCC. The prognostic significance of the number of removed lymph nodes in OCCC may differ depending on the FIGO stage.

Knockout of vasohibin-2 reduces tubulin carboxypeptidase activity and increases paclitaxel sensitivity in ovarian cancer.

Vasohibin-1 (VASH1) is a VEGF-inducible endothelium-derived angiogenesis inhibitor, and vasohibin-2 (VASH2), its homolog, exhibits proangiogenic activity. VASH2 is expressed by various cancer cells and accelerates tumor angiogenesis and progression. VASH2 was recently shown to exhibit tubulin carboxypeptidase (TCP) activity related to microtubule functions. Paclitaxel (PTX), an effective chemotherapeutic agent that is widely used to treat ovarian cancer, inhibits microtubule depolymerization and may interact with VASH2. We herein established several VASH2 knockout ovarian cancer cell lines using the CRISPR/Cas9 genome editing system to examine the intracellular tubulin detyrosination status and PTX chemosensitivity. The knockout of VASH2 did not affect the proliferation or sphere-forming activity of ovarian cancer cells in vitro. A Western blot analysis of VASH2 knockout cells revealed the weak expression of detyrosinated tubulin and upregulated expression of cyclin B1. The knockout of VASH2 significantly increased chemosensitivity to PTX, but not to cisplatin in ovarian cancer cell lines. The knockout of VASH2 reduced TCP activity and increased cyclin B1 expression, resulting in increased PTX chemosensitivity in ovarian cancer cells. The inhibition of angiogenesis and regulation of microtubule activity may be achieved in ovarian cancer treatment strategies targeting VASH2.

Efficacy and toxicity of pegylated liposomal doxorubicin as therapy for recurrent ovarian cancer in relation to the number of previous chemotherapy regimens: Comparison with gemcitabine.

AIM: Pegylated liposomal doxorubicin (PLD) is one of the second-line chemotherapy regimens for platinum-resistant recurrent ovarian cancer, but in clinical practice, it is also used for third or subsequent lines of chemotherapy. There is no report on the efficacy and toxicity of PLD in relation to the number of previous chemotherapy regimens. The purpose of this study was to clarify these points and compare with the results of gemcitabine (GEM) therapy we reported previously. METHODS: We retrospectively reviewed the medical records of patients with platinum-resistant recurrent ovarian cancer who underwent two or more cycles of PLD therapy between July 2009 and March 2017 at our institution. We used our reported data of GEM for comparison analysis. RESULTS: Seventy-eight patients were enrolled in this study. The overall response rate was 19.2% and the disease control rate (DCR) was 53.8%. The DCR with 1, 2, 3, and 4 or more previous regimens was 53.8%, 48.6%, 63.6% and 66.7%, respectively. Grade 3/4 neutropenia and anemia developed in 59.0% and 12.8%, respectively. Grade 2 or higher hand -foot syndrome, stomatitis, and liver dysfunction developed in 25.6%, 25.6% and 2.6%, respectively. When the number of previous regimens was 3 or higher, the DCR of PLD was significantly higher than that of GEM (64.7% vs 30.8%, P = 0.037). CONCLUSION: The DCR did not decrease with a greater number of previous regimens. When the number of previous regimens was 3 or higher, PLD therapy had a superior DCR to GEM therapy. Toxicity was tolerable in PLD therapy.

CRISPR/Cas9-mediated cervical cancer treatment targeting human papillomavirus E6.

High-risk human papillomavirus (HPV) is a common cause of cervical cancer. HPV E6 oncoprotein promotes the degradation of host tumor suppressor gene p53, leading to the development of tumors. Therapeutic strategies that specifically target E6, which is constitutively expressed in tumors and is not present in normal tissues, may be highly effective and safe. CRISPR-CRISPR associated protein 9 (Cas9) is one of the genome editing technologies that has recently garnered attention, and is used to knockout target gene expression. By combining cervical cancer cell lines engineered to constitutively express Cas9 and an adeno-associated virus (AAV) vector carrying a single guide (sg) RNA targeting E6 (AAV-sgE6), the present study sought to investigate the effects of this novel therapeutic approach on cervical cancer. The Cas9 gene was transfected into three high-risk HPV-positive cervical cancer cell lines (HeLa, HCS-2, and SKG-I) to establish cell lines that constitutively expressed Cas9. Using these cell lines, genetic mutations and their frequencies, as well as the levels of protein expression, apoptosis and cell proliferation were examined in vitro. In addition, the effects of AAV-sgE6 were examined in a mouse model of cervical cancer in vivo by a single administration of AAV-sgE6 directly into subcutaneous tumors. The results demonstrated that multiple mutations occurred frequently in the targeted E6 genomic sequence in cervical cancer cells transduced with AAV-sgE6. In addition, these AAV-sgE6-transduced cells had reduced expression of E6, increased expression of p53, increased apoptosis and their growth was suppressed in a concentration-dependent manner. Furthermore, subcutaneous tumor growth was significantly suppressed in vivo following intratumoral administration of AAV-sgE6, and adverse events due to AAV-sgE6 administration were not observed. Collectively, the present results indicated that targeting E6 expression in high-risk HPV by CRISPR-Cas9 is a highly specific and effective strategy that may be effective in treating patients with cervical cancer.

New criteria for the omission of lymphadenectomy in endometrioid carcinoma.

OBJECTIVE: To establish new criteria for the omission of lymphadenectomy in patients with endometrioid carcinoma. METHODS: We retrospectively reviewed 185 cases of histologically confirmed endometrioid carcinoma by hysterectomy at Jichi Medical University Hospital between January 2006 and December 2011. We reviewed patient medical records to detect risk factors for lymph node metastasis to identify the optimum criteria for lymphadenectomy omission. RESULTS: Univariate analysis revealed risk factors for lymph node metastasis to be a large tumor size (volume index ≥40 cm³) (p<0.0001), tumor diameter >2 cm (p=0.0003), myometrial invasion ≥50% based on pre-operative MRI (p=0.0366), elevated serum CA125 (pre-menopausal value ≥70 U/mL, post-menopausal value ≥25 U/mL) (p=0.0004), and lymphadenopathy on pre-operative CT scans (p=0.0002). Multivariate analysis indicated that tumor volume index, tumor diameter, elevated serum CA125, and CT scans positive for lymphadenopathy were independent risk factors for lymph node metastasis. Thus, we set tumor diameter >2 cm, elevated serum CA125, and CT scans positive for lymphadenopathy as risk factors. In cases with no risk factors, the rate of lymph node metastasis was 2.1%, which rose to 8.9%, 30.4%, and 58.3% for those with one, two, and three risk factors, respectively. The rate of para-aortic lymph node metastasis rose from 0% to 2.5%, 10.9%, and 41.7% among those with zero, one, two, and three risk factors, respectively. CONCLUSIONS: We propose that lymphadenectomy can be omitted in cases of endometrioid carcinoma that do not have any of the following risk factors: tumor diameter >2 cm, elevated serum CA125, and a CT scan positive for lymphadenopathy. We believe that these new criteria will limit inter-institutional differences as they are all objective factors. Further, they are useful in predicting lymph node metastasis, including para-aortic lymph node metastasis, based on the number of risk factors present.

Impact of the number of removed lymph nodes on recurrence-free survival in stage I ovarian clear cell carcinoma.

BACKGROUND: Although there have been several reports regarding the significance of staging lymphadenectomy for early stage ovarian clear cell carcinoma (CCC) patients, there have been few reports focusing on the number of removed lymph nodes. The aim of this study was to evaluate the impact of the number of removed lymph nodes on recurrence-free survival (RFS) in stage I ovarian CCC. METHODS: The subjects were patients with ovarian CCC who underwent surgery between January 1988 and December 2013. Clinicopathological variables were obtained from the medical records retrospectively. Statistical analysis using Kaplan-Meier method, log-rank test, and Cox proportional hazards model was performed. RESULTS: A total of 68 patients were entered into this study. The median number of removed lymph nodes was 56.5 (21-135). We calculated that the cutoff value of the number of removed lymph nodes for predicting recurrence was 35. RFS of the group with ≥ 35 removed lymph nodes was significantly better than that of the group with < 35 removed lymph nodes (p = 0.001). Similarly, RFS of stage IA and PS 0 or 1 was significantly better than that of stage IC (p = 0.029) and PS 2 or 3 (p = 0.001), respectively. Multivariate analysis revealed that the number of removed lymph nodes, stage, and PS was independent predictors for RFS. CONCLUSIONS: This study showed that the number of removed lymph nodes ≥ 35 was an independent predictor for improved RFS for stage I ovarian CCC. Sufficient lymphadenectomy may improve prognosis for stage I ovarian CCC.

Prognosis of endometrial cancer patients with and without symptoms at recurrence.

AIM: During routine follow-up for postoperative endometrial cancer, we have encountered patients with and without symptoms at recurrence. In this study, we investigated whether or not there is a difference in the prognosis between patients with and without symptoms at recurrence. METHODS: We reviewed endometrial cancer patients who had been treated in our hospital between 1998 and 2007. Routine follow-up was conducted by our facility criteria. We investigated recurrence-free survival (RFS), presence or absence of symptoms at recurrence, overall survival from recurrence (OSFR), and overall survival (OS). RESULTS: The subjects were 293 patients. Recurrence was detected in 46 patients. The median RFS was 15 (1-103) months. At the time of recurrence, symptoms were present in 14 patients and absent in 32 patients. In groups with and without symptoms at recurrence, the median OSFR were 36 (2-100) and 45 (2-139) months, respectively. The median OS were 55 (6-163) and 100 (11-178) months, respectively. There were no significant differences in either parameter. Independent prognostic factors for OSFR and OS were histopathologic types other than endometrioid carcinoma (vs endometrioid carcinoma, hazard ratio = 3.102 and 3.008, respectively) and RFS of 14 months or shorter (vs 15 months or longer, hazard ratio = 2.378 and 3.739, respectively). CONCLUSION: There was no difference in the prognosis between the groups with and without symptoms at recurrence. Independent prognostic factors of recurrent patients were histopathologic types and RFS. A large-scale study should be conducted to examine the necessity of routine follow-up for detecting recurrence in the absence of symptoms.