Prognostic value of endometriosis in patients with stage I ovarian clear cell carcinoma: Experiences at three academic institutions.

OBJECTIVES: To investigate the prognostic value of endometriosis in patients with stage I ovarian clear cell carcinoma (OCCC). METHODS: The medical records of patients with stage I OCCC who had undergone complete staging surgery followed by systemic chemotherapy were retrospectively reviewed. RESULTS: A total of 237 women were included in this study. Univariate analysis revealed that the patients with endometriosis-associated ovarian carcinoma (EAOC) had significantly improved recurrence-free survival (RFS) and overall survival (OS) than those without EAOC (5-year RFS: 91.4% vs. 73.0%, respectively, and 5-year OS: 97.5% vs. 89.9%). However, EAOC was not identified as a significant prognostic predictor in multivariate analysis. The potential risk factors determined to be associated with EAOC included the pretreatment CA-125 level, FIGO stage, lymphovascular space invasion (LVSI), and menopausal status (P<0.001, P=0.0031, P=0.020, and P=0.038, respectively). CONCLUSIONS: Endometriosis was not independently associated with the prognosis of the OCCC patients, even when the tumor was confined to stage I. However, the intrinsic relationship between endometriosis and OCCC warrants further investigation.

[Expression of C1QBP gene and its correlation with drug resistance in human resistance choriocarcinoma cell line].

OBJECTIVE: To examine the complement component 1 Q subcomponent-binding protein (C1QBP) gene expression in human resistance choriocarcinoma cell lines and its parental cell line JeG-3, and to investigate whether silence C1QBP by small interference RNA could reverse the resistance of human resistance choriocarcinoma cell lines to its relevant chemotherapy drugs. METHODS: Expression of C1QBP mRNA and protein in cells were detected by real- time fluorogenic quantitative PCR and western blot, respectively. The difference of C1QBP expression was compared between human resistance choriocarcinoma cell lines and its parental cell line JeG-3. Sub-cellular location was proved by confocal immunofluorescence microscopy. A lentiviral vector containing short hairpin RNA (shRNA) targeting C1QBP was constructed and cotransfected with the packaging plasmid mixture into 293T cells by lipofectamine 2000. The human resistance choriocarcinoma cell lines were infected with the packaged lentivirus. Real- time fluorogenic quantitative PCR and western blot were used to validate whether the C1QBP gene expression was silenced. The cell counting kit 8 (CCK8) was used to determine the drug sensitivity. RESULTS: (1) The C1QBP mRNA expression levels among four human resistance choriocarcinoma cell lines [JeG-3/floxuridiuum (FUDR), JeG- 3/methotrexate (MTX), JeG-3/etoposide (VP), JeG-3/dactinomycin (KSM)] were 2.520 ±0.680, 1.770 ± 0.230, 1.940 ± 0.090 and 1.740 ± 0.350 folds compared to that in JeG- 3 cells. The C1QBP protein was higher expression level in human resistance choriocarcinoma cell lines than that in JeG-3. The immunofluorescence methods and confocal analysis showed that C1QBP localized predominantly in the mitochondrial matrix. (2) The C1QBP mRNA expression in JeG-3/FUDR cells after infected with lentiviral vector were decreased by 93.1% (P < 0.01). The protein expression of C1QBP in JeG-3/FUDR cells after infected with lentiviral vector were almost completely suppressed. The resistance indexes of four human resistance choriocarcinoma cell lines (JeG-3/FUDR, JeG-3/MTX, JeG-3/VP, JeG-3/KSM) were respectively 86.3% , 93.9% , 92.8% and 89.9%, which were decreased remarkably by knockdown the C1QBP expression (P < 0.05). CONCLUSIONS: C1QBP is overexpressed in human resistance choriocarcinoma cell lines compared with parental cell line JeG-3. Inhibition of C1QBP by lentivirus- mediated small interference RNA could effectively reverses the resistance of human resistance choriocarcinoma cell lines to its relevant chemotherapy drugs.

Thymidylate synthase mRNA expression does not predict resistance to floxuridine in a choriocarcinoma cell line.

OBJECTIVE: Pyrimidine antagonist-based chemotherapy is a recommended chemotherapeutic treatment for gestational trophoblastic neoplasia in China. The main reason for treatment failure is chemoresistance. We established a floxuridine (FUDR)-resistant human choriocarcinoma JeG-3/FUDRA sub-line and investigated the role of thymidylate synthase (TS) transcript levels in chemoresistance prediction. STUDY DESIGN: The JeG-3/FUDRA sub-line was established by intermittent exposure to increasing doses of FUDR. Levels of TS transcripts were measured by real-time fluorescence quantitative reverse transcription-polymerase chain reaction. beta-hCG secretion in these cell lines has also been detected by using a chemoluminescence method. RESULTS: The JeG-3/FUDRA sub-line had a resistance index of 31.62. When exposed to 5.1 x 10(-7) M FUDR, the secretion of beta-hCG was enhanced dramatically, but with the increasing of the FUDR concentration, it was downregulated gradually. According to the concentration of FUDR exposure from low to high, the multiples of free beta-hCG secretion increased 13.19-, 4.76-, 1.90-, 1.44- and 0.47-fold, respectively. The TS transcript level was down-regulated by exposure to a low concentration of FUDR and then gradually increased with increasing doses of the drug. But when a certain concentration was reached, the expression would not increase but decrease sharply. By sub-lines of different concentration of FUDR exposure varying from low to high, the multiples of increases comparing the TS transcript level to parental cells were 0.56-, 0.42-, 1.02-, 2.78- and 2.06-fold, respectively. CONCLUSION: We successfully established an FUDR-resistant human choriocarcinoma sub-line and found that the expression of TS mRNA in FUDR-resistant JeG-3 cells was related to the concentration and phase of FUDR exposure. These data suggest that TS mRNA levels would be of limited use as biomarkers for the prediction of chemoresistance to FUDR-based chemotherapy.

Genotyping of a Chinese family with 46,XX and 46,XY 17-hydroxylase deficiency.

BACKGROUND: 17-Hydroxylase deficiency is a rare form of congenital adrenal hyperplasia caused by CYP17A1 gene mutations. METHOD: A 46,XY and a 46,XX Chinese patients with 17-hydroxylase deficiency in a family and their four generations family members were genotyped by PCR-sequencing method. RESULTS: Two CYP17 gene mutations were identified from these patients. Among them, IVS1-1G > A was a novel splicing mutation which disrupted the acceptor signal of exon 2 and might create a new exon after exon 1. The indel mutation of TAC329AA was a one-base deletion mutation and one-base change at codon 329 in exon 6. CONCLUSION: The results confirmed the diagnosis of 17-hydroxylase deficiency in these two patients and their autosome recessive heritage mode. The TAC329AA indel mutation had been identified in several reports of Chinese and Asian, suggesting that codon 329 was an unstable point of the CYP17 gene and this mutation was a prevalent CYP17 mutation in the Asian population. Although the noval mutation IVS1-1G > A founded in this family need more study to know its machinism of interrupting P450c17 function.

[Establishment of floxuridine-resistant JeG-3 subline and the role of thymidylate synthetase mRNA expression in chem-resistant-prediction.].

OBJECTIVE: To establish human choriocarcinoma JeG-3 cell line resistant to floxuridine (FUDR) and describe the characteristics of this FUDR-resistant subline. The thymidylate synthase (TS) expression level in FUDR-resistant subline was also discussed. METHODS: The FUDR-resistant sub-line JeG-3/FUDRA was established by intermitted exposure to grads increased FUDR. Reversed microscope was used to observe the morphological changes in FUDR-resistant sub-line. Population doubling time was calculated and compared based on the growth curve of these two cell lines, cell cycles and chromosomal ploidy were assayed with flow cytometry methods. The chemo-luminescence assay was used to detect the hormone secretion by two kinds of cell lines. The resistant index (RI) was measured by cell counting kit-8 (CCK-8) assay. Quantitative RT-PCR was used to detect the mRNA expression level of TS and we also detected the TS mRNA expression level in different doses exposed subline. RESULTS: The RI of JeG-3/FUDRA was 31.62. Compared with the JeG-3 cell, the FUDR-resistant cell line had gross changes in morphological, cell growth, cell cycles and chromosomal numbers. The ability of human chorionic gonadotrop (hCG) and progesterone secretion was lower in JeG-3/FUDRA subline. The trend of TS mRNA expression was: while exposed to low concentration of FUDR, the TS mRNA expression level was downregulated, then followed the increasing dose of the drug, the expression level of TS mRNA ascended gradually. When the terminal concentration was reached, the expression level of TS mRNA in JeG-3/FUDRA subline was higher than that of JeG-3 cell line (P < 0.05). CONCLUSIONS: We established the FUDR-resistant subline of JeG-3 successfully. The TS mRNA expression level is stage-related to the different concentration and different phase in FUDR exposure. Our data suggested that TS mRNA expression level may not be used as a biomarker to predict the chemosensitivity in FUDR-based chemotherapy.

A potential mechanism of breakthrough bleeding associated with progestin: involvement in alteration of endometrial endothelial cells.

OBJECTIVE: To explore the potential mechanism of breakthrough bleeding associated with progestin with in vitro methods. METHODS: The isolation and culture of human endometrial endothelial cells (HEECs) was performed with the method established in our laboratory. The content and activity of urokinase-type plasminogen activator (uPA) and the content of plasminogen activator inhibitor-1 (PAI-1) in cell supernatants after incubated with different concentrations of progesterone (0-5 micromol/L) and 17beta-estradiol (0, 0.1, or 1 nmol/L) were measured by method of ELISA. Apoptosis rate of HEECs was measured by flow cytometry. Viable cell count was measured by MTT. RESULTS: The increased level of progesterone (0.5-5 micromol/L) combined with 17beta-estradiol elevated content and activity of uPA while the production of PAI-1 remained unchanged. The apoptosis of HEECs was inhibited along with the increment of total viable cell counts at higher concentrations of progesterone with 17beta-estradiol. CONCLUSION: The inhibition of apoptosis and increased content and activity of uPA may contribute to the occurrence of irregular bleeding associated with progestin use to some extent

Genistein inhibits proliferation of human endometrial endothelial cell in vitro.

OBJECTIVE: To explore the effect of genistein on proliferation of human endometrial endothelial cells (HEECs) and glandular epithelium. METHODS: In vitro HEECs and human endometrial cancer-1B cell (HEC-1B) were cultured with 0, 1, 10, 50, 100, and 200 micromol/L of genistein alone or indicated concentrations of genistein combined with 0.2 or 1 nmol/L 17beta-estradiol (17beta-E2). Cell proliferation was determined by [3H]-thymidine incorporation and cell cycle was measured by flow cytometry. RESULTS: After 96 hours of treatment, genistein inhibited the proliferation of HEECs in a dose-dependent manner. The stimulation index reduced from 100% (without genistein treatment) to about 1% (200 micromol/L genistein). HEECs were arrested at G1/0 and G2/M phase when treated with genistein for 96 hours. When the concentration of genistein was 200 micromol/L, the percentages of HEECs at G1/0, G2/M, and S phase were 96.0%, 2.1%, and 1.9%, respectively. However, when HEECs were treated without genistein, the percentages of HEECs at G1/0, G2/M, and S phase were 76.7%, 8.5%, and 14.7%, respectively. 17beta-E2 could not influence the effects of genistein on the proliferation of HEECs. Meanwhile, genistein could suppress the proliferation of HEC-1B. If the stimulation index of HEC-1B was defined as 100% when HEC-1B was treated with different doses of 17beta-E2 (without genistein), it was 67%, 19%, as well as 32% when cell was supplemented with 200 micromol/L genistein combined with 0, 0.2, or 1 nmol/L 17beta-E2, respectively. CONCLUSION: Genistein at the concentration of 200 micromol/L can sufficiently inhibit the proliferation of HEECs and endometrial glandular epithelium simultaneously in vitro.

[Incomplete P450 17 alpha enzyme deficiency: report of six cases].

OBJECTIVES: To summarize the characteristics, differential diagnosis and management of incomplete 17 alpha-hydroxylase/17, 20-lyase deficiency (17 OHD) of Chinese patients. METHODS: Six cases of incomplete 17 OHD from Peking Union Medical College Hospital were studied retrospectively through analyzing their clinical data, and the molecular pathogenic mechanism was discussed after literature review. RESULTS: Four cases of 46, XX incomplete 17 OHD were reported. The clinical characteristics included female phenotype, various degrees of breast development and absent or sparse axillary/pubic hair, oligomenorrhea or secondary amenorrhea, recurrent luteinized ovarian cysts, hypogonadism with persistent hyperprogesteronemia or high serum 17 alpha-hydroxyprogesterone level, with or without hypokalemic hypertension. There were also 2 cases of 46, XY incomplete 17 OHD, in which ambiguous genitalia were present besides hypokalemic hypertension. CONCLUSIONS: Incomplete 17 OHD is a very rare form of congenital enzymatic deficiencies of steroid synthesis, which should be included in the differential diagnosis when there are menstrual disorders, sexual infantilism, recurrent ovarian cysts or ambiguous genitalia. Under such circumstances, hyperprogesteronemia offers a valuable clue for further investigation.

Salvage Chemotherapy for Patients With Recurrent or Persistent Ovarian Clear Cell Carcinoma: A Retrospective Study of 164 Cases.

The purpose of this study was to evaluate the effects of salvage chemotherapy on recurrent or persistent ovarian clear cell carcinoma (CCC) with the goal of identifying a more rational treatment regimen for this lethal disease.The medical records of patients with CCC were retrospectively reviewed to select patients that were subsequently treated for recurrent or persistent disease.Of the 164 women with recurrent or persistent CCC, 485 chemotherapy courses with 1766 cycles were administered. Overall, the clinical benefit rate (CBR) was 39.4%, and the mean progression-free survival (PFS) was 4.5 months. Grade 3/4 toxicities occurred in 94 courses (19.4%). The CBR for TC was 45.1%, with a PFS of 3.7 months. Compared to that of TC, the CBRs for PC and CC were significantly lower (P = 0.020 and 0.021, respectively). The CBRs and PFS for PAF-C were slightly higher (P = 0.518 and 0.077, respectively), but showed a significantly higher adverse event rate (AER, P = 0.039). The CBR for bevacizumab was 50% with an extraordinarily long PFS (49.8 months). Gemcitabine and oxaliplatin had similar values for CBRs (44.4% and 44.1%) and PFS (2.5 and 3.4 months), respectively. Docetaxel (weekly) exhibited a notably low AER of 2.7%, and topotecan was associated with a relatively long PFS (7.7 months).For cis/carboplatin-pretreated patients, the existing active agents, such as oxaliplatin, gemcitabine, topotecan, and especially bevacizumab, are promising. Docetaxel (weekly) is well tolerated and might offer a particularly viable option for heavily pretreated patients. However, additional research to identify for a continued search for the optimal combination of chemotherapeutics or novel agents is still warranted.

Diagnostic value of the neutrophil-to-lymphocyte ratio and the combination of serum CA-125 for stages III and IV endometriosis.

BACKGROUND: Currently, all the diagnostic indicators for endometriosis lack perfect sensitivity and specificity. According to the characteristic of endometriosis, we analyzed the new biomarker neutrophil-to-lymphocyte ratio (NLR) and the combination of NLR and serum CA-125 to investigate their diagnostic value for identifying stages III and IV endometriosis. METHODS: The values of serum CA-125 and routine blood tests were collected from 197 patients with endometriosis, 102 with benign tumors and 112 healthy individuals. We investigated the sensitivity and specificity of NLR and its combination with serum-CA-125 for diagnosing stages III and IV endometriosis by using receiver operating characteristic (ROC). RESULTS: The mean values of NLR, the combination of serum CA-125 and NLR (combination) of the groups with stages III and IV endometriosis were significantly higher than the other two groups. Serum CA-125, NLR, and the combined biomarkers could significantly discriminate the stages III and IV endometriosis group from the other two groups (P < 0.05). NLR shows a lower sensitivity of 57.9% and specificity of 65.2% with a cutoff value at 1.82. And the combination of biomarkers has the highest AUC of 0.949 with a sensitivity of 86.8% and specificity of 92.0% at the cutoff value of 44.40. In addition, for patients with negative CA-125, 55.36% and 53.57% of the patients were able to be diagnosed with endometriosis by using NLR alone and the combination of biomarkers. CONCLUSION: For diagnosing stages III and IV endometriosis, the neutrophil-to-lymphocyte ratio is a better adjuvant to serum CA-125, and the neutrophil-to-lymphocyte ratio is valuable in diagnosing stages III and IV endometriosis for patients with negative serum CA-125.

Elevated levels of gremlin-1 in eutopic endometrium and peripheral serum in patients with endometriosis.

OBJECTIVE: To examine the expression of gremlin-1 (GREM1) on the levels of messenger RNA (mRNA) and protein in eutopic endometrium and its serum level in patients with endometriosis. DESIGN: Prospective, experimental study using reverse-transcription polymerase chain reaction, Western blot, immunofluorescence, and ELISA. SETTING: Gynecological oncology laboratory in a department of obstetrics and gynecology in a medical college in China. PATIENT(S): Thirty-five patients with endometriosis and 23 healthy control women. INTERVENTION(S): During surgery, the eutopic endometria and peripheral serum were obtained from the patients with endometriosis and the control women. MAIN OUTCOME MEASURE(S): The cellular compartment location of GREM1 expression was examined by using immunofluorescent double staining. The expression levels of mRNA and protein for GREM1 were determined by reverse-transcription polymerase chain reaction and Western blot, respectively. The serum level of GREM1 was measured by indirect ELISA. RESULT(S): The expression of GREM1 was defined within endometrial blood vessel endothelium exclusively, with the concomitant expressions of GREM1 and CD146. The expression of GREM1 on the levels of mRNA and protein was significantly higher in eutopic endometria of patients with endometriosis than in those from healthy control women. According to the ELISA established in our laboratory, the concentration of GREM1 in peripheral serum that was collected during the follicular menstrual phase of patients with endometriosis was significantly higher than that in serum from healthy control women. CONCLUSION(S): Gremlin-1 plays a role to some extent in the aberrant angiogenesis of eutopic endometrium in patients with endometriosis. It is possible that the peripheral serum level of GREM1 is a prospective serum biomarker of endometriosis.