Solid tumours showing oligoprogression to immune checkpoint inhibitors have the potential for abscopal effects.

PURPOSE: Given the uncertainty surrounding the abscopal effect (AE), it is imperative to identify promising treatment targets. In this study, we aimed to explore the incidence of AE when administering radiotherapy to patients with oligoprogressive solid tumours while they are undergoing treatment with immune checkpoint inhibitors (ICIs). MATERIALS AND METHODS: In this multicentre prospective observational study, oligoprogressive disease was defined as a < 20% increase in lesions compared to > 2 months before enrolment. We enrolled patients who requested radiotherapy during the ICI rest period between 2020 and 2023. AE was considered present if ≥ 1 non-irradiated lesion decreased by ≥ 30% before the next line of systemic therapy started. RESULTS: Twelve patients were included in this study; the common primary lesions were in the lungs (four patients) and kidneys (three patients). AEs were observed in six (50%) patients, with a median time to onset of 4 (range 2-9) months after radiotherapy. No significant predictors of AEs were identified. Patients in the AE group had a significantly better 1-year progression-free survival (PFS) rate than those in the non-AE group (p = 0.008). Two patients from the AE group were untreated and progression-free at the last follow-up. Four (33%) patients experienced grade 2 toxicity, with two cases attributed to radiotherapy and the other two to ICI treatment. No grade 3 or higher toxicities were observed in any category. CONCLUSION: Patients with oligoprogressive disease may be promising targets with potential for AEs. AEs can lead to improved PFS and, in rare cases, to a certain progression-free period without treatment. Irradiating solid tumours in patients with oligoprogressive disease during immune checkpoint inhibitor therapy may be a promising target with the potential for abscopal effects (AEs). AEs can lead to improved progression-free survival and, in rare cases, to a certain progression-free period without treatment.

Impact of Chronic Exposure to Endometriosis on Perinatal Outcomes: Establishment of a Mouse Model.

The purpose of this study was to establish a new mouse model of endometriosis that mimics real-world women's health problems, in which women continue to be affected by endometriosis long before they wish to become pregnant, and to evaluate the impact of "chronic exposure to endometriosis" on perinatal outcome. Endometriosis was established by the intraperitoneal injection of homologous minced mouse uteri. Vehicle was injected for the control. Mating was initiated either 1 or 43 days after disease establishment (Young or Aged studies, respectively). Mice were sacrificed on 18 dpc. The number pups and resorptions were counted and pups' body weights (BW) were measured, and the endometriosis lesion was identified and weighted. In the Young study, the number of resorptions and BW were comparable between the groups. In the Aged study, the number of resorptions was significantly higher and BW was significantly lower in endometriosis than that in control. The total weight of endometriosis lesion per dam was significantly lower in the Aged compared to the Young endometriosis group; however, not a single mouse was found to have any lesions at all. These results suggest that in addition to the presence of endometriosis per se, "chronic exposure to endometriosis" prior to pregnancy affect perinatal outcomes.

Establishment of a novel mouse model of adenomyosis suitable for longitudinal and quantitative analysis and perinatal outcome studies.

The purpose of this study was to establish a novel mouse model of adenomyosis suitable for longitudinal and quantitative analyses and perinatal outcome studies. Using a 30 G needle, the entire uterine wall of one horn was mechanically punctured at a frequency of 100 times/1 cm (adenomyosis horn). The other horn was left unpunctured (control horn). Balb/c mice were sacrificed on day 14 (D14) or day 65 (D65) (n = 3 each). The uterus was fixed, paraffin-embedded, sliced, and stained. Lesions were detected and counted, and their volumes were measured. Cell proliferation and fibrosis were assessed by Ki67 and Masson's Trichrome staining, respectively. Blood vessels were detected using CD31 immunostaining. Some of the mice (n = 4), were mated and the date of delivery, litter size, number of implantations, and number and volume of postpartum lesions were measured. The number of lesions per horn did not differ between D14 and D65. The volume of the entire lesion was significantly greater on D65 than on D14 (p < 0.0001). The volume of the epithelial part of the lesion was significantly greater in D65 (p < 0.0001). The volume of the stromal part of the lesion was also greater on D65 (p < 0.0001). The percentage of Ki67 positive cells in the epithelial part of the lesion was significantly higher on D14 (p < 0.05). In contrast, the percentage of Ki67-positive cells in the stromal part was significantly higher on D65 (p < 0.01). Vascular density in the lesions was higher in on D65 (p < 0.05). The percentage of fibrotic area was significantly higher on D65 (p < 0.01). The date of delivery was slightly earlier than that reported for healthy mice of the same strain. The litter size was smaller than that reported in previous research. The number of implantation sites did not differ between the control and the adenomyosis horn. The number and volume of lesions did not differ between the non-pregnant and postpartum groups. This model can be applied to evaluate the pathogenesis of adenomyosis, validate the efficacy of therapeutic agents, and evaluate the effect of adenomyosis on pregnancy and vice versa.

Automatic one-click planning for hippocampal-avoidance whole-brain irradiation in RayStation.

This study aimed to verify the accuracy of auto-contouring and auto-dose optimization for hippocampal-avoidance whole-brain radiation therapy (HA-WBRT). Head computed tomography (CT) images of 15 patients were selected. The regions of interest, containing the brain, hippocampus, eyes, and lacrimal glands, were contoured manually and automatically on CT images. They were compared and evaluated for concordance rates using the Simpson coefficient. To verify the performance of dose optimization, auto-dose planning was compared with manual planning for 15 cases. All optimization plans were performed using the volumetric modulated arc therapy technique. The automatically contoured brain showed a very high concordance rate with the manually contoured brain; the Simpson coefficient was 0.990 ± 0.01. Contrastingly, the concordance rate of the hippocampal contour was low at 0.642 ± 0.15 (right) and 0.500 ± 0.16 (left); however, the rate improved to 0.871 ± 0.09 (right) and 0.852 ± 0.11 (left) with an additional 3-mm margin. For 2% of each planning target volume with the prescribed dose (D2%) and Dmean, there was no significant difference between the automatic and manual plans (35.50 Gy vs 35.23 Gy; p = 0.233 and 33.09 Gy vs 32.84 Gy; p = 0.073, respectively). The D98% was significantly better for the manual plan than for the automatic plan (25.49 Gy vs 26.11 Gy; p < 0.01). Dmax and D100% for the hippocampus did not show any significant difference between the automatic and manual plans (15.65, 16.09 Gy (right, left) vs 15.51, 15.80 Gy; p = 0.804, 0.233 and 8.08, 8.03 Gy vs 8.13, 8.01 Gy; p = 0.495, 1 respectively). The accuracy of auto-contouring for HA-WBRT can be guaranteed by providing an appropriate margin, and the precision of the auto-dose optimization was comparable to that of the manual plan.

The roles of polymorphonuclear myeloid-derived suppressor cells in endometriosis.

OBJECTIVES: This study aimed to determine the systemic and local proportions, focal localization, and characteristics of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) in endometriosis. STUDY DESIGN: Peripheral blood and peritoneal fluid were obtained from patients with a benign gynecologic condition (controls) or endometriosis. PMN-MDSCs were defined as CD33+HLA-DRlow/-CD14-CD15+ and monocytic (M)-MDSCs were defined as CD33+HLA-DRlow/-CD14+CD15-, and were identified using flowcytometry. Ovarian endometriotic tissues were obtained, and the expression of lectin-type oxidized low density lipoprotein receptor-1 (LOX1) as a marker of PMN-MDSCs, arginine 1 (Arg1), and matrix metalloproteinase 9 (MMP9) were detected using immunohistochemistry. Anti-Ly6G antibody was administered to endometriosis model mice, and the number and weight of the lesions were measured, and cell proliferations and apoptosis in the lesions were analyzed using Ki67 immunohistochemistry and TUNEL assay. RESULTS: In the peripheral blood, the proportion of PMN-MDSCs was significantly higher in endometriosis (3.20 vs 1.63 %, p < 0.05), but the proportion of M-MDSCs did not differ between the groups. In the peritoneal fluid, the proportion of PMN-MDSCs was significantly higher in endometriosis (7.82 × 10-1% vs 6.48 × 10-2%, p < 0.05), whereas the proportion of M-MDSCs did not differ between the groups. PMN-MDSCs were detected in the stromal cell layer of the endometriotic cyst wall. Double staining for LOX1 and Arg1, and LOX1 and MMP9 was confirmed. Administration of Ly6G antibody did not change the number or weight of endometriosis lesions, but significantly decreased Ki67-positive cells and increased TUNEL-positive cells in the lesions. CONCLUSIONS: PMN-MDSCs may contribute to the pathogenesis of endometriosis via Arg1 and MMP9 expression.

Stereotactic body radiation therapy for Japanese patients with localized prostate cancer: 2-year results and predictive factors for acute genitourinary toxicities.

OBJECTIVE: We aimed to report the 2-year results of stereotactic body radiation therapy for prostate cancer and identify the clinical and dosimetric factors that predict acute genitourinary toxicities. METHODS: We retrospectively reviewed the medical records of patients with non-metastatic prostate cancer treated at Toyota Memorial Hospital between 2017 and 2020. The patients were treated with stereotactic body radiation therapy with a total dose of 36.25 Gy in five fractions on consecutive weekdays. While low-risk patients received radiotherapy alone, intermediate- to high-risk patients also received androgen deprivation therapy. RESULTS: We analysed a total of 104 patients, including 10, 60 and 34 low-, intermediate- and high-risk patients, respectively. The median follow-up duration was 2 years. We did not observe biochemical/clinical recurrence, distant metastasis or death from prostate cancer. One patient died of another cause. Grade 2 acute genitourinary toxicity was observed in 40 (38%) patients. Age (P = 0.021), genitourinary toxicity of grade ≥1 at baseline (P = 0.023) and bladder mean dose (P = 0.047) were significantly associated with the incidence of grade 2 acute genitourinary toxicity. The cut-off value of 65 years for age and 10.3 Gy for the bladder mean dose were considered the most appropriate. Grade 2 acute gastrointestinal toxicity was observed in five (5%) patients. None of the patients experienced grade ≥3 acute or late toxicity. CONCLUSIONS: Stereotactic body radiation therapy is feasible for Japanese patients with prostate cancer, with acceptable acute toxicity. Age, genitourinary toxicity at baseline and bladder mean dose predict grade 2 acute genitourinary toxicity.

Use of selective PGE2 receptor antagonists on human endometriotic stromal cells and peritoneal macrophages.

Non-hormonal therapeutic strategies for endometriosis are needed. The aim of this study was to characterize the effects of prostaglandin (PG)E2 receptor inhibitors to explore their potential as novel therapeutic strategies for endometriosis. The expression of PGE2 receptors (EP2 and EP4) in donated tissues from human ovarian endometriosis, adenomyosis and peritoneal endometriosis was examined using immunohistochemistry. Human endometriotic stromal cells (ESC) isolated from ovarian endometriotic tissue and peritoneal macrophages were treated with EP2 and EP4 antagonists. cAMP accumulation and the effect of EP antagonists were measured using cAMP assays. DNA synthesis in ESC was detected using bromodeoxyuridine incorporation analysis. Interleukin (IL)-6 and IL-8 protein levels in ESC supernatants were measured using ELISAs. mRNA expression level for aromatase by ESC, and selected cytokines by peritoneal macrophages was measured using RT-PCR. EP2 and EP4 receptors were expressed in cells derived from control and diseased tissue, ovarian endometriotic, adenomyotic and peritoneal lesions. A selective EP2 antagonist reduced DNA synthesis, cAMP accumulation and IL-1ß-induced proinflammatory cytokine secretion and aromatase expression. A selective EP4 antagonist negated IL-1ß-induced IL-6 secretion and aromatase expression. In peritoneal macrophages, EP expression was elevated in endometriosis samples but the EP4 antagonist reduced cAMP levels and expression of vascular endothelial growth factor, chemokine ligand 2 and chemokine ligand 3 mRNA. EP2 and EP4 are functioning in endometriosis lesions and peritoneal macrophages, and their selective antagonists can reduce EP-mediated actions, therefore, the EP antagonists are potential therapeutic agents for controlling endometriosis.

Role of high-dose salvage radiotherapy for oligometastases of the localised abdominal/pelvic lymph nodes: a retrospective study.

BACKGROUND: Abdominal/pelvic lymph node (LN) oligometastasis, a pattern of treatment failure, is observed occasionally, and radiotherapy may work as salvage therapy. The optimal prescription dose, however, is yet to be determined. This study assessed the efficacy of high-dose radiotherapy. METHODS: The medical records of 113 patients at 4 institutes were retrospectively analysed who had 1 to 5 abdominal/pelvic LN oligometastases and were treated with definitive radiotherapy between 2008 and 2018. The exclusion criteria included non-epithelial tumours, uncontrolled primary lesions, palliative intent, and re-irradiation. The prescription dose was evaluated by using the equivalent dose in 2 Gy fractions (EQD2). Patients receiving EQD2 ≥ 60 Gy were placed into the high-dose group, and the remaining others the low-dose group. Kaplan-Meier analyses were performed to evaluate overall survival (OS), local control (LC), and progression-free survival (PFS). Univariate log-rank and multivariate Cox proportional hazards model analyses were performed to explore predictive factors. Adverse events were compared between the high-dose and low-dose groups. RESULTS: The primary tumour sites included the colorectum (n = 28), uterine cervix (n = 27), endometrium (n = 15), and ovaries (n = 10). The rate of 2-year OS was 63.1%, that of LC 59.7%, and that of PFS 19.4%. On multivariate analyses, OS were significantly associated with solitary oligometastasis (hazard ratio [HR]: 0.48, p = 0.02), LC with high-dose radiotherapy (HR: 0.93, p < 0.001), and PFS with long disease-free interval (HR: 0.59, p = 0.01). Whereas high-dose radiotherapy did not significantly improve 2-year OS in the entire cohort (74.8% in the high-dose vs. 52.7% in the low-dose; p = 0.08), it did in the subgroup of solitary oligometastasis (88.8% in the high-dose vs. 56.3% in the low-dose; p = 0.009). As for Late grade ≥ 3 adverse event, ileus was observed in 7 patients (6%) and gastrointestinal bleeding in 4 (4%). No significant association between the irradiation dose and adverse event incidence was found. CONCLUSIONS: As salvage therapy, high-dose radiotherapy was recommendable for oligometastasis in the abdominal/pelvic LNs. For solitary oligometastasis, LC and OS were significantly better in the high-dose group.

PAX8: A Highly Sensitive Marker for the Glands in Extragenital Endometriosis.

In cases of extragenital endometriosis or microscopic endometriosis lesions, pathological diagnosis can be challenging because endometriotic stroma and glands represent only a minor component of fibrotic endometriotic lesions. For better accuracy of diagnosis, the development of a sensitive and specific epithelial marker is beneficial. Previous studies showed that PAX8 is a highly sensitive and specific marker for primary and metastatic Mullerian epithelial tumors. Therefore, we sought to examine whether PAX8 is a highly sensitive marker for glands in extragenital endometriosis. Eight and 47 samples of ovarian endometrioma and extragenital endometriosis, respectively, were evaluated in this study. We calculated the percentage of samples positively immunostained for PAX8, CD10, estrogen receptor (ER), and progesterone receptor (PR). PAX8 was positive for endometriotic epithelial cells in 95.7% (45/47) of extragenital endometrioses and in 100% (8/8) of ovarian endometrioses. CD10 was positive for endometriotic stromal cells in 97.9% (46/47) of extragenital endometrioses. PAX8 was strongly positive for glands, even in a CD10-negative case. The expression of PAX8, CD10, and PR was not affected by preoperative hormonal therapy, and the positive rate of ER staining was significantly reduced by preoperative hormonal therapy. In conclusion, PAX8 is a highly sensitive epithelial marker for extragenital endometriosis. This specific expression was maintained under hormonal therapy. It is noteworthy that extragenital endometriosis maintains the expression of this lineage marker, although it occurs at various sites, and its cause and mechanism of development might be different. PAX8 nuclear expression can be useful in detecting extragenital endometriosis in clinical practice.

IFITM1 is a Novel, Highly Sensitive Marker for Endometriotic Stromal Cells in Ovarian and Extragenital Endometriosis.

When the presence of endometriotic lesions are not evident by hematoxylin and eosin staining, CD10 is used to highlight and confirm the presence of endometriotic stroma. However, CD10 is not specific only to the endometrial stroma but is also expressed in many other cells. Recently, interferon-inducible transmembrane protein 1 (IFITM1) was reported as a highly specific immunohistochemical marker of normal endometrial stroma and endometrial stromal neoplasm. In this study, we examined the expression of IFITM1 and CD10 in 18 cases of ovarian endometriosis and 44 cases of extragenital endometriosis. Among the 62 patients, 62 (100.0%) were positive for IFITM1 and 60 (96.8%) for CD10, and CD10 was negative in 2 cases that were positive for IFITM1. Additionally, we found that IFITM1 sensitivity was unaffected by the presence or absence of hormonal therapy. To the best of our knowledge, this represents the first demonstration of IFITM1 as a highly sensitive stromal marker of ovarian and extragenital endometriosis.

The effects of tokishakuyakusan, a traditional Japanese medicine (kampo), ferulic acid and paeoniflorin, on human endometriotic stromal cells and peritoneal macrophages.

OBJECTIVES: To evaluate the anti-inflammatory and anti-angiogenetic effects of Tokishakuyakusan (TSS), a traditional Japanese medicine (Kampo), and its ingredients, ferulic acid (FA) and paeoniflorin (PA) on endometriotic stromal cells (ESC) and peritoneal macrophages. STUDY DESIGN: Endometriotic tissues were obtained from 16 patients and peritoneal macrophages were obtained from 11 patients that had undergone laparoscopic surgery for ovarian endometriosis. ESC isolated from endometriotic tissues and peritoneal macrophages were cultured, and pre-treated with 300 µg/mL of TSS, 500 µM FA or 50 µM PA. ESC and peritoneal macrophages were then stimulated with IL-1ß. Concentrations of IL-8 and VEGF protein in supernatants were then detected and measured using specific ELISAs. TSS (4 g/kg body weight) was orally administered to female Sprague-Dawley rats. The concentration of FA in plasma and uteri was measured using liquid chromatography-mass spectrometry with tandem mass spectrometry (LC-MS/MS).　 RESULTS: TSS and FA but not PA decreased the secretion of inflammatory cytokine (IL-8) and angiogenic factor (VEGF) in ESC. TSS and FA also suppressed the secretion of inflammatory cytokine (IL-8) from peritoneal macrophages. FA was detected in plasma and in uterine tissues after the oral administration of TSS to rats. CONCLUSIONS: Our study demonstrates that TSS has anti-inflammatory and anti-angiogenic effects on endometriosis related cells by controlling inflammatory cytokine and growth factor secretion from cells, and these effects, at least partially, may be due to the direct effects of the TSS ingredient FA.

Activation of endoplasmic reticulum stress mediates oxidative stress-induced apoptosis of granulosa cells in ovaries affected by endometrioma.

Endometriosis exerts detrimental effects on ovarian physiology and compromises follicular health. Granulosa cells from patients with endometriosis are characterized by increased apoptosis, as well as high oxidative stress. Endoplasmic reticulum (ER) stress, a local factor closely associated with oxidative stress, has emerged as a critical regulator of ovarian function. We hypothesized that ER stress is activated by high oxidative stress in granulosa cells in ovaries with endometrioma and that this mediates oxidative stress-induced apoptosis. Human granulosa-lutein cells (GLCs) from patients with endometrioma expressed high levels of mRNAs associated with the unfolded protein response (UPR). In addition, the levels of phosphorylated ER stress sensor proteins, inositol-requiring enzyme 1 (IRE1) and double-stranded RNA-activated protein kinase-like ER kinase (PERK), were elevated in granulosa cells from patients with endometrioma. Given that ER stress results in phosphorylation of ER stress sensor proteins and induces UPR factors, these findings indicate that these cells were under ER stress. H2O2, an inducer of oxidative stress, increased expression of UPR-associated mRNAs in cultured human GLCs, and this effect was abrogated by pretreatment with tauroursodeoxycholic acid (TUDCA), an ER stress inhibitor in clinical use. Treatment with H2O2 increased apoptosis and the activity of the pro-apoptotic factors caspase-8 and caspase-3, both of which were attenuated by TUDCA. Our findings suggest that activated ER stress induced by high oxidative stress in granulosa cells in ovaries with endometrioma mediates apoptosis of these cells, leading to ovarian dysfunction in patients with endometriosis.

Endometriosis Triggers Excessive Activation of Primordial Follicles via PI3K-PTEN-Akt-Foxo3 Pathway.

CONTEXT: The ovarian reserve is reduced in patients with endometriosis. We hypothesize that the phosphatidylinositol 3-kinase (PI3K)-phosphatase and tensin homolog deleted on chromosome 10 (PTEN) Akt-Forkhead box O (Foxo3) pathway is involved in reducing the ovarian reserve. OBJECTIVE: To elucidate the signaling mechanism by which endometriosis decreases ovarian reserve. DESIGN: Studies were conducted by using a mouse model for endometriosis and human ovaries. The endometriosis mouse model was established and ammonium trichloro (dioxoethylene-o,o') tellurate (AS101), an inhibitor of PI3K-PTEN-Akt pathway, was administered to experimental mice. Human ovaries were collected during surgery from patients with endometrioma or from patients with no ovarian pathology (control ovaries). The number of follicles and expression of Foxo3, PTEN, phosphorylated mammalian target of rapamycin and phosphorylated Akt by oocytes in primordial follicles in mouse and human ovaries were detected by immunohistochemical staining and evaluated. RESULTS: In the endometriosis mouse model, the proportion of primordial follicles was diminished, and the proportion of primary, secondary, antral, and growing follicles was increased in comparison with controls. In both mouse and human ovaries, the PI3K-PTEN-Akt-Foxo3 pathway was activated in samples from endometriosis. Administration of AS101 restored the proportion of primordial follicles in endometriotic mice ovaries to control levels. CONCLUSIONS: The current study describes the excessive activation of primordial follicles and the role of the PI3K-PTEN-Akt-Foxo3 pathway in the reduction of ovarian reserve associated with endometriosis. Our results suggest that a PI3K-PTEN-Akt inhibitor should be considered for further investigation as promising medicines for the prevention of the ovarian reserve reduction in patients with endometriosis.

IFITM1 is a Novel, Highly Sensitive Marker for Endometriotic Stromal Cells in Ovarian and Extragenital Endometriosis.

When the presence of endometriotic lesions are not evident by hematoxylin and eosin staining, CD10 is used to highlight and confirm the presence of endometriotic stroma. However, CD10 is not specific only to the endometrial stroma but is also expressed in many other cells. Recently, interferon-inducible transmembrane protein 1 (IFITM1) was reported as a highly specific immunohistochemical marker of normal endometrial stroma and endometrial stromal neoplasm. In this study, we examined the expression of IFITM1 and CD10 in 18 cases of ovarian endometriosis and 44 cases of extragenital endometriosis. Among the 62 patients, 62 (100.0%) were positive for IFITM1 and 60 (96.8%) for CD10, and CD10 was negative in 2 cases that were positive for IFITM1. Additionally, we found that IFITM1 sensitivity was unaffected by the presence or absence of hormonal therapy. To the best of our knowledge, this represents the first demonstration of IFITM1 as a highly sensitive stromal marker of ovarian and extragenital endometriosis.

Predictive factors of prognosis after radiation and steroid pulse therapy in thyroid eye disease.

To identify predictive factors of prognosis after radiotherapy with concurrent steroid pulse therapy for thyroid eye disease, retrospective analyses were performed among 77 patients. Clinical activity score and magnetic resonance imaging were used to evaluate degrees of orbital inflammation. As a pre-treatment work-up, the thyroid-stimulating antibody (TSAb) level was measured. During a median follow-up of 25.0 months, the 2-year cumulative relapse-free rate (CRFR) was 80.9%. In the univariate analysis, a worse 2-year CRFR was significantly associated with the presence of optic neuropathy (P = 0.001), a higher TSAb rate (P = 0.001), and lower standard deviation (SD) of signal intensity at the extraocular muscle in T2-weighted images (P = 0.006). In the multivariate analysis, TSAb rate and SD affected the CRFR independently. When TSAb activity of 2800% was set as a cut-off at 2 years after treatment, the predictive sensitivity and specificity of relapse were 81.2% and 90.6%, respectively. With regard to SD, the respective sensitivity and specificity values were 81.2% and 82.7% when 100 was set as a cut-off. In conclusion, high TSAb and low SD were significant risk factors for cumulative relapse in orbital radiotherapy. Cut-off values of 2800% for TSAb and 100 for SD may be suitable.

PAX8: A Highly Sensitive Marker for the Glands in Extragenital Endometriosis.

In cases of extragenital endometriosis or microscopic endometriosis lesions, pathological diagnosis can be challenging because endometriotic stroma and glands represent only a minor component of fibrotic endometriotic lesions. For better accuracy of diagnosis, the development of a sensitive and specific epithelial marker is beneficial. Previous studies showed that PAX8 is a highly sensitive and specific marker for primary and metastatic Mullerian epithelial tumors. Therefore, we sought to examine whether PAX8 is a highly sensitive marker for glands in extragenital endometriosis. Eight and 47 samples of ovarian endometrioma and extragenital endometriosis, respectively, were evaluated in this study. We calculated the percentage of samples positively immunostained for PAX8, CD10, estrogen receptor (ER), and progesterone receptor (PR). PAX8 was positive for endometriotic epithelial cells in 95.7% (45/47) of extragenital endometrioses and in 100% (8/8) of ovarian endometrioses. CD10 was positive for endometriotic stromal cells in 97.9% (46/47) of extragenital endometrioses. PAX8 was strongly positive for glands, even in a CD10-negative case. The expression of PAX8, CD10, and PR was not affected by preoperative hormonal therapy, and the positive rate of ER staining was significantly reduced by preoperative hormonal therapy. In conclusion, PAX8 is a highly sensitive epithelial marker for extragenital endometriosis. This specific expression was maintained under hormonal therapy. It is noteworthy that extragenital endometriosis maintains the expression of this lineage marker, although it occurs at various sites, and its cause and mechanism of development might be different. PAX8 nuclear expression can be useful in detecting extragenital endometriosis in clinical practice.

Expression of Nerve Injury-Induced Protein1 (Ninj1) in Endometriosis.

OBJECTIVE: The aim of this study was to clarify the expression of Ninj1 in endometriosis and adenomyosis lesions, and its inductive factor in human endometriotic stromal cells (ESCs). BACKGROUND: Nerve injury-induced protein 1 (Ninj1) is a molecule originally identified in dorsal root ganglion neurons and Schwann cells after nerve injury and promotes neurite outgrowth. The aim of this study was to clarify the expression of Ninj1 in endometriosis and adenomyosis lesions, and its inductive factor in human endometriotic stromal cells (ESCs). MATERIALS AND METHODS: Tissues were obtained with consent from patients diagnosed with ovarian endometrioma (n = 15 in total), peritoneal endometriosis (n = 5), adenomyosis (n = 5), and other gynecological disorders (n = 5, control) during surgery. Immunohistochemistry was conducted in order to detect Ninj1 protein expression in the lesion of endometriosis, adenomyosis, and eutopic endometrium. Nerve fibers in the ovarian endometrioma were detected by positive staining of PGP-9.5. To evaluate the effects of IL-1ß on Ninj1 gene expression in endometriosis, ESCs isolated from ovarian endometrioma (n = 5) were treated with IL-1ß (5 ng/mL) for 3 or 6 hours. Messenger RNA (mRNA) expression for Ninj1 was examined using quantitative RT-PCR. RESULTS: The Ninj1 protein was expressed by ovarian endometrioma, peritoneal endometriotic, and adenomyotic tissue. Nerve fibers were found in the areas of positive staining for Ninj1 in ovarian endometrioma. IL-1ß, an indicator of inflammation in endometriosis, significantly increased Ninj1 mRNA expression by ESC. CONCLUSION: Our study demonstrates that Ninj1 is expressed in endometriosis and adenomyosis and is induced by the inflammatory stimuli. Given the neurogenetic property of Ninj1, our results imply that Ninj1, induced by inflammation in endometriosis lesion, may contribute to the pathogenesis of pain symptoms characteristic of endometriosis.

Involvement of immune cells in the pathogenesis of endometriosis.

Endometriosis is characterized by the implantation and growth of endometriotic tissues outside the uterus. It is widely accepted the theory that endometriosis is caused by the implantation of endometrial tissue from retrograde menstruation; however, retrograde menstruation occurs in almost all women and other factors are required for the establishment of endometriosis, such as cell survival, cell invasion, angiogenesis, and cell growth. Immune factors in the local environment may, therefore, contribute to the formation and progression of endometriosis. Current evidence supports the involvement of immune cells in the pathogenesis of endometriosis. Peritoneal neutrophils and macrophages secrete biochemical factors that help endometriotic cell growth and invasion, and angiogenesis. Peritoneal macrophages and NK cells in endometriosis have limited capability of eliminating endometrial cells in the peritoneal cavity. An imbalance of T cell subsets leads to aberrant cytokine secretions and inflammation that results in the growth of endometriosis lesions. It is still uncertain whether these immune cells have a role in the initial cause and/or stimulate actions that enhance disease; however, in either case, modulating the actions of these cells may prevent initiation or disease progression. Further studies are needed to deepen the understanding of the pathology of endometriosis and to develop novel management approaches of benefit to women suffering from this disease.

Drospirenone reduces inflammatory cytokines, vascular endothelial growth factor (VEGF) and nerve growth factor (NGF) expression in human endometriotic stromal cells.

Drospirenone has been used as a progestin in oral contraceptives with ethinyl estradiol (DRSP/EE) and is expected to regulate endometriosis, however, the direct effects of drospirenone on endometriosis have not been clarified. The aim of this study was to evaluate the anti-inflammatory, anti-angiogenic and anti-neurogenic effects of drospirenone on endometriotic stromal cells (ESC). ESC isolated from endometriotic tissues were obtained from patients during laparoscopic surgery for ovarian endometriosis. ESC were exposed to IL-1ß and cultured in the absence or presence of drospirenone. mRNA expression was evaluated using quantitative RT-PCR, and protein was measured using ELISAs. To evaluate the effect of drospirenone on progesterone receptor (PR) and mineralocorticoid receptor (MR), ESC were transfected with siRNA against PR (siPR) and MR (siMR), and cultured in the presence or absence of drospirenone. Drospirenone significantly decreased IL-6, IL-8, VEGF and NGF mRNA expression by ESC. Drospirenone (10-5M) significantly decreased IL-6 secretion and 10-7M drospirenone decreased IL-8 and VEGF secretion. Knockdown of PR, but not MR, negated the effects of drospirenone. In summary, this study demonstrates that drospirenone has anti-inflammatory, anti-angiogenic and anti-neurogenic effects on ESC and these effects are mediated by PR. These drospirenone effects may contribute to the regulatory effects of drospirenone-containing oral contraceptives on endometriosis.

Dienogest reduces proliferation, NGF expression and nerve fiber density in human adenomyosis.

OBJECTIVES: To evaluate the in vivo effect of dienogest on proliferation, apoptosis, aromatase expression, vascular density, nerve growth factor (NGF) expression and nerve fiber density in human adenomyosis tissue. STUDY DESIGN: Twelve women who underwent hysterectomy for adenomyosis were enrolled. Six patients received dienogest treatment prior to hysterectomy (dienogest group), and age-matched six patients who had not received any hormonal treatment for ≥3 months before surgery (control group). Cell proliferation, vascular and nerve fiber density in adenomyosis tissue were evaluated by staining for Ki67, von Willebrand factor and PGP9.5, respectively. Apoptosis was detected using the TUNEL assay. The expression aromatase and NGF were evaluated by staining for corresponding antibodies. RESULTS: The proportion of Ki67 positive epithelial cells was significantly lower in samples from dienogest-treated patients in comparison with controls (p<0.05). The density of blood vessels in adenomyosis was marginally lower in the dienogest group in comparison with controls but statistical significance was not reached (p=0.07). The intensity of NGF expression and the density of nerve fibers were significantly lower in the dienogest group compared with controls (p<0.05 for both). CONCLUSION: This study demonstrates that adenomyosis, taken from patients treated with dienogest, shows remarkable histological features, such as reductions in proliferation, NGF expression and nerve fiber density. These findings indicate the impact of dienogest on local histological events, and explains its therapeutic effect on adenomyosis.