Neutrophil recruitment and function in endometriosis patients and a syngeneic murine model.

Endometriosis is a chronic inflammatory, gynecological disease characterized by the presence of endometrial-like tissue lesions outside of the uterus. Neutrophils are elevated in the systemic circulation and peritoneal fluid of endometriosis patients; however, whether and how neutrophils contribute to endometriosis pathophysiology remain poorly understood. With emerging roles for neutrophils in chronic and sterile inflammatory conditions, we sought to provide in-depth characterization of neutrophil involvement in endometriosis. We demonstrate that neutrophils reside within patient endometriotic lesions and that patient lesions possess a microenvironment that may influence neutrophil recruitment and function. We also provide the first evidence that systemic circulating neutrophils from endometriosis patients display distinct transcriptomic differences compared neutrophils from healthy control subjects. Time course characterization of our syngeneic, immunocompetent mouse model of endometriosis revealed that neutrophils are rapidly recruited to the peritoneal environment early after endometriotic lesion establishment and remain present in murine lesions long term. In vivo neutrophil depletion altered the systemic and peritoneal immune microenvironment of mice with endometriosis as demonstrated by changes in pro-inflammatory and angiogenic mediators. Taken together, these findings highlight a novel role for neutrophils in early events such as angiogenesis and modulation of the local inflammatory environment associated with endometriosis pathogenesis.

Rasch analysis of The Shoulder Pain and Disability Index (SPADI) in a postrepair rotator cuff sample.

STUDY DESIGN: Clinical measurement study: Level of evidence (N/A) INTRODUCTION: The Shoulder Pain and Disability Index (SPADI) is a self-reported outcome measure of pain and disability related to shoulder pathology. In comparison to Classical Test Theory (CTT), Rasch analysis offers a more rigorous examination of the measurement properties of a scale. PURPOSE OF THE STUDY: This study utilizes Rasch analysis to evaluate the psychometric properties of the SPADI to propose potential modifications and avenues for future investigation. METHODS: SPADI scores (n = 212) from participants one-year post rotator cuff repair were collected from an outpatient specialty clinic. Fit to the Rasch model, unidimensionality of the subscales, and areas of bias were evaluated. RESULTS: Both the pain and disability subscales satisfied the requirements of the Rasch model with very minimal modifications and demonstrated unidimensionality. The person separation index was found to be high (P > .80), indicating reliability and internal consistency. Sex and the affected dominant side influenced how people scored on the SPADI (Differential item functioning (DIF)). CONCLUSIONS: The findings suggest some patients in our sample have difficulty discriminating between item responses, particularly within the middle of the scale. Rasch analysis supports the clinical measurement properties of consistency and reliability, previously determined by CTT methods.

Mucinous metaplasia of the endometrium: current concepts.

OBJECTIVES: The objective of this review was to discuss the highly variable terminology describing mucinous metaplasia of the endometrium, histologic features and characteristic findings by special stains and immunohistochemistry, differential diagnostic challenges, current treatment and prognosis. METHODS: A review of the literature was performed on mucinous metaplasia of the endometrium. RESULTS: Mucinous metaplasia is an uncommon type of endometrial epithelial metaplasia, often associated with hyperestrogenic states or hormone replacement therapy. Histologic findings range from simple endocervical-type mucinous epithelium to architecturally complex mucinous proliferations. Mucinous metaplasia often presents a significant diagnostic challenge in endometrial biopsies and should be differentiated from endocervical microglandular hyperplasia, and mucinous or mixed mucinous and endometrioid carcinomas. Simple mucinous metaplasia is believed to have a low risk of associated carcinoma, whereas architecturally complex cases often show invasive malignancy on subsequent biopsy. CONCLUSIONS: Complex mucinous proliferations should be managed with dilatation and curettage and repeat biopsy within 6months, and strong consideration should be given to a hysterectomy in persistent mucinous proliferations.

Human Papillomavirus-Related Ovarian Metastasis With Endocervical Adenocarcinoma: Report of 2 Cases and Review of Literature.

BACKGROUND: Most endocervical adenocarcinomas are associated with human papillomavirus (HPV) infection. Studies suggest that synchronous endocervical and ovarian tumors can contain identical HPV subtypes and that, in this setting, the ovarian tumors likely represent metastases from the endocervical adenocarcinoma rather than 2 independent primaries. However, there are still relatively few reports in the literature. RESULTS: We describe 2 patients with HPV-related endocervical adenocarcinoma or adenocarcinoma in situ who had metastatic ovarian tumors that simulated primary ovarian neoplasms. After total hysterectomy and bilateral salpingo-oophorectomy, patient 1, in her mid-40s, was diagnosed with endocervical adenocarcinoma in situ and patient 2, in her early 50s, was diagnosed with endocervical adenocarcinoma showing early focal stromal invasion. The ovarian tumors in both cases simulated independent borderline mucinous tumors without evidence of surface involvement or spread beyond the ovary. However, in both cases, the cervical and ovarian tumors showed diffuse, strong P16 staining and contained identical high-risk HPV subtypes (subtypes 16 and 18 in patient 1 and subtype 16 in patient 2). Patient 1 was treated with radiation therapy and remains recurrence free 5 years after diagnosis, and patient 2 has recently completed combined modality treatment with radiation therapy and cisplatin chemotherapy and remains recurrence free 10 months after diagnosis. CONCLUSIONS: A high index of suspicion and ancillary testing, including P16 immunostaining and molecular genetic testing for HPV, is required to properly diagnose and subclassify HPV-related endocervical adenocarcinoma metastatic to the ovary.

Identification of the IGF1/PI3K/NF κB/ERK gene signalling networks associated with chemotherapy resistance and treatment response in high-grade serous epithelial ovarian cancer.

BACKGROUND: Resistance to platinum-based chemotherapy remains a major impediment in the treatment of serous epithelial ovarian cancer. The objective of this study was to use gene expression profiling to delineate major deregulated pathways and biomarkers associated with the development of intrinsic chemotherapy resistance upon exposure to standard first-line therapy for ovarian cancer. METHODS: The study cohort comprised 28 patients divided into two groups based on their varying sensitivity to first-line chemotherapy using progression free survival (PFS) as a surrogate of response. All 28 patients had advanced stage, high-grade serous ovarian cancer, and were treated with standard platinum-based chemotherapy. Twelve patient tumours demonstrating relative resistance to platinum chemotherapy corresponding to shorter PFS (< eight months) were compared to sixteen tumours from platinum-sensitive patients (PFS > eighteen months). Whole transcriptome profiling was performed using an Affymetrix high-resolution microarray platform to permit global comparisons of gene expression profiles between tumours from the resistant group and the sensitive group. RESULTS: Microarray data analysis revealed a set of 204 discriminating genes possessing expression levels which could influence differential chemotherapy response between the two groups. Robust statistical testing was then performed which eliminated a dependence on the normalization algorithm employed, producing a restricted list of differentially regulated genes, and which found IGF1 to be the most strongly differentially expressed gene. Pathway analysis, based on the list of 204 genes, revealed enrichment in genes primarily involved in the IGF1/PI3K/NF κB/ERK gene signalling networks. CONCLUSIONS: This study has identified pathway specific prognostic biomarkers possibly underlying a differential chemotherapy response in patients undergoing standard platinum-based treatment of serous epithelial ovarian cancer. In addition, our results provide a pathway context for further experimental validations, and the findings are a significant step towards future therapeutic interventions.

DICER1 Mutation in Recurrent Ovarian Sertoli-Leydig Cell Tumor: A Case Report.

BACKGROUND: DICER1 mutation has been linked to development of Sertoli-Leydig cell tumor and cystic nephroma, among other neoplasms. CASE: We present a unique case of recurrent ovarian Sertoli-Leydig cell tumor in a pediatric patient with a known DICER1 mutation and history of cystic nephroma. She underwent surgical staging and adjuvant chemotherapy, and her recurrences have been treated with chemotherapy, whole-abdomen radiation therapy, and further surgical debulking. CONCLUSION: This report adds to the small body of evidence about this rare but unexpectedly highly aggressive tumor, especially in the recurrent setting, and reminds the reader of the importance of cancer diagnosis in this population.

The dysregulation of leukemia inhibitory factor and its implications for endometriosis pathophysiology.

Endometriosis is an estrogen dominant, chronic inflammatory disease characterized by the growth of endometrial-like tissue outside of the uterus. The most common symptoms experienced by patients include manifestations of chronic pelvic pain- such as pain with urination, menstruation, or defecation, and infertility. Alterations to Leukemia Inhibitory Factor (LIF), a cytokine produced by the luminal and glandular epithelium of the endometrium that is imperative for successful pregnancy, have been postulated to contribute to infertility. Conditions such as recurrent implantation failure, unexplained infertility, and infertility associated diseases such as adenomyosis and endometriosis, have demonstrated reduced LIF production in the endometrium of infertile patients compared to fertile counterparts. While this highlights the potential involvement of LIF in infertility, LIF is a multifaceted cytokine which plays additional roles in the maintenance of cell stemness and immunomodulation. Thus, we sought to explore the implications of LIF production within ectopic lesions on endometriosis pathophysiology. Through immunohistochemistry of an endometrioma tissue microarray and ELISA of tissue protein extract and peritoneal fluid samples, we identify LIF protein expression in the ectopic lesion microenvironment. Targeted RT qPCR for LIF and associated signaling transcripts, identify LIF to be significantly downregulated in the ectopic tissue compared to eutopic and control while its receptor, LIFR, is upregulated, highlighting a discordance in ectopic protein and mRNA LIF expression. In vitro treatment of endometriosis representative cell lines (12Z and hESC) with LIF increased production of immune-recruiting cytokines (MCP-1, MCP-3) and the angiogenic factor, VEGF, as well as stimulated tube formation in human umbilical vein endothelial cells (HUVECs). Finally, LIF treatment in a syngeneic mouse model of endometriosis induced both local and peripheral alterations to immune cell phenotypes, ultimately reducing immunoregulatory CD206+ small peritoneal macrophages and T regulatory cells. These findings suggest that LIF is present in the ectopic lesions of endometriosis patients and could be contributing to lesion vascularization and immunomodulation.

EMT transcription factors snail and slug directly contribute to cisplatin resistance in ovarian cancer.

BACKGROUND: The epithelial to mesenchymal transition (EMT) is a molecular process through which an epithelial cell undergoes transdifferentiation into a mesenchymal phenotype. The role of EMT in embryogenesis is well-characterized and increasing evidence suggests that elements of the transition may be important in other processes, including metastasis and drug resistance in various different cancers. METHODS: Agilent 4 × 44 K whole human genome arrays and selected reaction monitoring mass spectrometry were used to investigate mRNA and protein expression in A2780 cisplatin sensitive and resistant cell lines. Invasion and migration were assessed using Boyden chamber assays. Gene knockdown of snail and slug was done using targeted siRNA. Clinical relevance of the EMT pathway was assessed in a cohort of primary ovarian tumours using data from Affymetrix GeneChip Human Genome U133 plus 2.0 arrays. RESULTS: Morphological and phenotypic hallmarks of EMT were identified in the chemoresistant cells. Subsequent gene expression profiling revealed upregulation of EMT-related transcription factors including snail, slug, twist2 and zeb2. Proteomic analysis demonstrated up regulation of Snail and Slug as well as the mesenchymal marker Vimentin, and down regulation of E-cadherin, an epithelial marker. By reducing expression of snail and slug, the mesenchymal phenotype was largely reversed and cells were resensitized to cisplatin. Finally, gene expression data from primary tumours mirrored the finding that an EMT-like pathway is activated in resistant tumours relative to sensitive tumours, suggesting that the involvement of this transition may not be limited to in vitro drug effects. CONCLUSIONS: This work strongly suggests that genes associated with EMT may play a significant role in cisplatin resistance in ovarian cancer, therefore potentially leading to the development of predictive biomarkers of drug response or novel therapeutic strategies for overcoming drug resistance.

Implications of dysregulated endogenous cannabinoid family members in the pathophysiology of endometriosis.

OBJECTIVE: To determine the involvement of the endocannabinoid (EC) family member in the pathophysiology of endometriosis (EMS). DESIGN: Mass spectrometry analysis of plasma and tissue samples from patients with EMS, controls, and a mouse model of EMS and messenger RNA and immunohistochemistry analysis of the samples from patients with EMS and controls. SETTING: Academic teaching hospital and university. PATIENT(S): Patients with EMS and healthy fertile control subjects. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Endocannabinoid analysis in patient plasma, EMS lesions, and healthy endometrial samples. RESULT(S): Circulating ECs were detected in the plasma samples, whereas no significant changes were observed in patients with EMS compared with healthy fertile controls. However, the palmitoylethanolamide levels were significantly higher in the EMS lesions than in the endometrium from patients with EMS. Similarly, genes involved in the EC signaling pathways were differentially expressed in the EMS lesions. Analysis of cannabinoid 1 and 2 receptors in the EMS lesions revealed a significantly lower cannabinoid 2 receptor expression, whereas no significant changes were observed in cannabinoid 1 receptor expression compared with those in the endometrium from both patients with EMS and healthy fertile controls. The palmitoylethanolamide levels were significantly elevated in plasma from EMS mice compared with that from sham controls and in EMS lesions compared with uterine samples. CONCLUSION(S): Together, we provide evidence toward dysregulation of members of the ECs in both patients with EMS and the mouse model of EMS. These findings will advance the knowledge of the role of ECs in EMS and their potential implications as therapeutic targets.

Uterine Tumor Resembling Ovarian Sex Cord Tumor: A Distinct Entity Characterized by Recurrent NCOA2/3 Gene Fusions.

Uterine tumor resembling ovarian sex-cord tumor (UTROSCT) is a rare and distinctive neoplasm of unclear histogenesis, and uncertain malignant potential. These neoplasms morphologically resemble sex-cord stromal tumors of the ovary, and possess a polyphenotypic immunophenotype. Their molecular pathogenesis has yet to be elucidated; notably, however, tumors lack alterations found in other uterine tumors bearing sex-cord-like differentiation, such as endometrial stromal sarcoma. Following identification of an index patient with an ESR1-NCOA3 fusion gene by RNA-sequencing, we undertook a retrospective review for additional cases of UTROSCT. We identified a total of 4 patients, with an average age of 53 years (range, 38 to 68 y). RNA-sequencing was performed in all cases, revealing an ESR1-NCOA3 fusion in 2 cases and one case each with related ESR1-NCOA2 and GREB1-NCOA2 fusions. Each of the tumors showed histologic and an immunophenotype features within the previously reported spectrum of UTROSCT; interestingly, one case contained prominent spindle cell fascicles and another was largely comprised of sheets of small round cells. Our results demonstrate UTROSCT are defined by recurrent fusions involving NCOA2 or NCOA3, a finding that is directly amenable to diagnostic evaluation. This study confirms UTROSCT is molecularly distinct from endometrial stromal sarcoma, and raises intriguing new questions into the pathogenesis of these neoplasms and possible relationship with other NCOA fusion-positive uterine tumors.

STAT1-associated intratumoural TH1 immunity predicts chemotherapy resistance in high-grade serous ovarian cancer.

High-grade serous ovarian carcinoma (HGSC) accounts for 70% of all epithelial ovarian cancers but clinical management is challenged by a lack of accurate prognostic and predictive biomarkers of chemotherapy response. This study evaluated the role of Signal Transducer and Activator of Transcription 1 (STAT1) as an independent prognostic and predictive biomarker and its correlation with intratumoural CD8+ T cells in a second independent biomarker validation study. Tumour STAT1 expression and intratumoural CD8+ T cell infiltration were assessed by immunohistochemistry as a multicentre validation study conducted on 734 chemotherapy-naïve HGSCs. NanoString-based profiling was performed to correlate expression of STAT1 target genes CXCL9, CXCL10 and CXCL11 with CD8A transcript expression in 143 primary tumours. Multiplexed cytokine analysis of pre-treatment plasma from resistant and sensitive patients was performed to assess systemic levels of STAT1-induced cytokines. STAT1 was validated as a prognostic and predictive biomarker in both univariate and multivariate models and its expression correlated significantly with intra-epithelial CD8+ T cell infiltration in HGSC. STAT1 levels increased the prognostic and predictive value of intratumoural CD8+ T cells, confirming their synergistic role as biomarkers in HGSC. In addition, expression of STAT1 target genes (CXCL9, CXCL10 and CXCL11) correlated significantly with levels of, and CD8A transcripts from intratumoural CD8+ T cells within the resistant and sensitive tumours. Our findings provide compelling evidence that high levels of STAT1, STAT1-induced chemokines and CD8+ T cells correlate with improved chemotherapy response in HGSC. These results identify STAT1 and its target genes as novel biomarkers of chemosensitivity in HGSC. These findings provide new translational opportunities for patient stratification for immunotherapies based on emerging biomarkers of inflammation in HGSC. An improved understanding of the role of interferon-inducible genes will be foundational for developing immunomodulatory therapies in ovarian cancer.

Blocking of stromal cell-derived factor-1 reduces neoangiogenesis in human endometriosis lesions in a mouse model.

PROBLEM: Endometriosis affects 5-10% of women and is characterized by the growth of endometrial tissue outside of the uterus. Establishing new blood supply is a fundamental requirement for endometriosis lesion growth. Endothelial progenitor cells (EPCs), recruited by stromal cell-derived factor-1 (SDF-1), contribute to neoangiogenesis in endometriotic lesions. We hypothesized that SDF-1 is central to the neoangiogenesis and survival of endometriotic lesions, and blocking of SDF-1 will reduce vascularization of lesions in a mouse model. METHOD OF STUDY: Using immunohistochemistry, we evaluated SDF-1 and CD34(+) EPCs in human endometriotic lesions and normal endometrium samples. EPCs were co-localized using CD34 and VEGFR2. Effects of SDF-1 blocking on endometriotic lesion survival were assessed in BALB/c-Rag2(-/-) /IL2rγ(-/-) mice engrafted with human endometrium and treated with SDF-1-blocking antibody or an isotype control. Weekly blood samples from experimental mice were analyzed for cytokines and EPCs. RESULTS: SDF-1 and CD34(+) EPCs were abundant in human endometriotic lesions compared with eutopic endometrium. In our mouse model, SDF-1-blocking antibody reduced CD31(+) microvessels compared with isotype control. CONCLUSION: Blocking SDF-1 reduces neovascularization and survival of lesions in a mouse model of endometriosis.

Differential Immunoreactivity of p16 in leiomyosarcomas and leiomyoma variants.

Several studies have now examined the cDNA expression profiles of healthy myometrium, leiomyomas (LM), and leiomyosarcomas (LMS). This has produced a list of candidate genes that might be useful tools for distinguishing these entities from each other. The potential candidates identified from this body of research include insulinlike growth factor 1, h-caldesmon, cytokeratin 18, and the cyclin-dependent kinase 4 inhibitor, p16. To determine whether the immunohistochemical expression of these proteins could aid in the diagnosis of LMS and LM variants, we constructed a tissue microarray consisting of cases of healthy myometrium (n = 10), LM (not otherwise specified and variants; n = 47), and LMS (n = 8), and then measured the immunoreactivity of each of these proteins. The cases were scored on the basis of staining intensity (weak, moderate, or strong) and extent (focal or diffuse) and were assigned a final score from 0 to +3. Immunostaining for p16 was statistically stronger in LMS than in LM and its subtypes (P < 0.001). Specifically, the p16 immunostaining score in LMS cases (n = 8) was at least +2, whereas the p16 immunostaining scores in all LM cases (n = 47) were either 0 (n = 35) or +1 (n = 12). The expression of the remaining antibodies did not show a statistically significant difference between the 2 groups. Furthermore, none of the markers studied showed any differences among the LM variants. The results of this study confirm the overexpression of p16 in LMS and suggest that p16 can serve as a reliable immunohistochemical marker in distinguishing uterine LMS from LM and its benign variants.