Annukka Pasanen

Integrated data analysis reveals uterine leiomyoma subtypes with distinct driver pathways and biomarkers.

Significance The clinical and scientific community widely regards uterine leiomyomas as a single entity, although evidence of genetic heterogeneity exists. The aim of this study was to explore transcriptional differences between leiomyomas harboring different genetic alterations, including high mobility group AT-hook 2 rearrangements, mediator complex subunit 12 mutations, biallelic inactivation of fumarate hydratase, and collagen, type IV, alpha 5-collagen, type IV, alpha 6 deletions. The evidence presented herein strongly suggests that specific driver mutations are the major determinants of expression changes in leiomyomas. Here we highlight subtype-specific expression differences in key driver pathways and emphasize the utility of stratification in leiomyoma research. Finally, we offer a set of candidate biomarkers that will facilitate the molecular classification of leiomyomas. Uterine leiomyomas are common benign smooth muscle tumors that impose a major burden on women’s health. Recent sequencing studies have revealed recurrent and mutually exclusive mutations in leiomyomas, suggesting the involvement of molecularly distinct pathways. In this study, we explored transcriptional differences among leiomyomas harboring different genetic drivers, including high mobility group AT-hook 2 (HMGA2) rearrangements, mediator complex subunit 12 (MED12) mutations, biallelic inactivation of fumarate hydratase (FH), and collagen, type IV, alpha 5 and collagen, type IV, alpha 6 (COL4A5-COL4A6) deletions. We also explored the transcriptional consequences of 7q22, 22q, and 1p deletions, aiming to identify possible target genes. We investigated 94 leiomyomas and 60 corresponding myometrial tissues using exon arrays, whole genome sequencing, and SNP arrays. This integrative approach revealed subtype-specific expression changes in key driver pathways, including Wnt/β-catenin, Prolactin, and insulin-like growth factor (IGF)1 signaling. Leiomyomas with HMGA2 aberrations displayed highly significant up-regulation of the proto-oncogene pleomorphic adenoma gene 1 (PLAG1), suggesting that HMGA2 promotes tumorigenesis through PLAG1 activation. This was supported by the identification of genetic PLAG1 alterations resulting in expression signatures as seen in leiomyomas with HMGA2 aberrations. RAD51 paralog B (RAD51B), the preferential translocation partner of HMGA2, was up-regulated in MED12 mutant lesions, suggesting a role for this gene in the genesis of leiomyomas. FH-deficient leiomyomas were uniquely characterized by activation of nuclear factor erythroid 2-related factor 2 (NRF2) target genes, supporting the hypothesis that accumulation of fumarate leads to activation of the oncogenic transcription factor NRF2. This study emphasizes the need for molecular stratification in leiomyoma research and possibly in clinical practice as well. Further research is needed to determine whether the candidate biomarkers presented herein can provide guidance for managing the millions of patients affected by these lesions.

Risk-stratification of endometrial carcinomas revisited: A combined preoperative and intraoperative scoring system for a reliable prediction of an advanced disease

OBJECTIVE The aim of this study is to develop a risk-scoring system for predicting lymph node and distant metastasis in endometrial carcinoma. METHODS A total of 1166 patients, treated at a single institution between 1-2007 and 12-2013, were included in the study. The association of stage IIIC-IV disease with demographic factors (age, body mass index), biochemical factors (complete blood count, serum CA125), preoperative histology and tumor size was examined in unadjusted analyses. Logistic regression analysis was used for the identification of variables that independently predict an advanced disease. RESULTS Thrombocytosis, serum CA125>35 U/mL, preoperative high-risk histology (nonendometrioid or grade 3 endometrioid carcinoma) and tumor size≥3 cm were independently associated with an advanced disease. These predictors were internally validated by a bootstrapping method with statistical significance. A risk-scoring system with an area under the receiver operating characteristic curve of 0.852 (95% confidence interval, 0.821-0.883) was developed. Total risk score points ranged from 0 to 8 for individual patients. With a cut-off of 1 point, stratifying 66.3% of patients to surgical staging, the model predicted stage IIIC-IV carcinomas with a sensitivity of 100%, specificity of 38.0%, positive predictive value of 17.1%, and negative predictive value of 100%. With the same cut-off, the corresponding values were 100%, 34.7%, 16.5% and 100% in predicting stage IIIC carcinomas in a subgroup that underwent lymphadenectomy and had a stage I-IIIC disease. CONCLUSIONS This risk-scoring system is highly sensitive in predicting an advanced stage endometrial carcinoma at a cut-off of risk score points that is associated with an acceptable lymphadenectomy rate.

Multiple clinical characteristics separate MED12-mutation-positive and -negative uterine leiomyomas

Up to 86% of uterine leiomyomas harbour somatic mutations in mediator complex subunit 12 (MED12). These mutations have been associated with conventional histology, smaller tumour size, and larger number of tumours within the uterus. Prior studies, with limited sample sizes, have failed to detect associations between other clinical features and MED12 mutations. Here, we prospectively collected 763 uterine leiomyomas and the corresponding normal myometrial tissue from 244 hysterectomy patients, recorded tumour characteristics, collected clinical data from medical records, and screened the tissue samples for MED12 mutations to assess potential associations between clinical variables and mutation status. Out of 763 leiomyomas, 599 (79%) harboured a MED12 mutation. In the analysis of tumour characteristics, positive MED12-mutation status was significantly associated with smaller tumour size, conventional histology, and subserous location, relative to intramural. In the analysis of clinical variables, the number of MED12-mutation-positive tumours showed an inverse association with parity, and the number of mutation-negative tumours showed a positive association with a history of pelvic inflammatory disease. This study confirmed the previously reported differences and discovered novel differentiating features for MED12-mutation-positive and -negative leiomyomas. These findings emphasise the relevance of specific driver mutations in genesis and presentation of uterine leiomyomas.

L1 Cell Adhesion Molecule as a Predictor of Disease-Specific Survival and Patterns of Relapse in Endometrial Cancer.

OBJECTIVE The aim was to study the association of L1 cell adhesion molecule (L1CAM) expression with the outcome of patients with endometrial cancer, especially with regard to conventional risk variables, and to compare the patterns of relapse in L1CAM-positive and -negative cancers. METHODS This was a retrospective study of 805 women. The Kaplan-Meier method and univariate and multivariate Cox regression models were applied for survival analyses. Missing data were replaced using multiple imputation. The median follow-up time was 51 months (range, 1-98). RESULTS One hundred twenty-one (15.0%) cases were L1CAM positive. L1CAM positivity was associated with high stage (I vs II-IV) (odds ratio [OR], 2.3), lymph node involvement (OR, 2.9), poor differentiation (OR, 6.1), non-endometrioid histology (OR, 9.9), lymphovascular space invasion (OR, 2.8), cervical stromal invasion (OR, 1.8), positive peritoneal cytology (OR, 4.1), and age older than 65 years (OR, 2.8). The frequencies of deep myometrial invasion (50% or deeper), tumor size 2 cm or greater, and body mass index 30 kg/m2 or greater were not significantly different between L1CAM-positive and -negative cases. L1CAM predicted poor disease-specific survival in endometrioid (P < 0.0001) but not in non-endometrioid carcinomas (P = 0.934). The negative impact of L1CAM on outcome was confirmed in a Cox multivariate disease-specific survival analysis. Univariate survival analyses in the different ESMO-ESGO-ESTRO endometrial cancer risk groups showed an association between L1CAM positivity and poor outcome in intermediate (hazard ratio, 12) and high-risk advanced metastatic (hazard ratio, 2.0) groups. Extra-abdominal relapses were more frequent in L1CAM-positive (13.2%) than L1CAM-negative (1.9%) stage I endometrioid carcinomas (P < 0.0001), whereas other site-specific relapses in local cancers were L1CAM independent. CONCLUSIONS L1CAM is associated with the occurrence of poor prognostic variables and predicts advanced disease in endometrial cancer. L1CAM predicts extra-abdominal relapses and poor survival in endometrioid endometrial cancer, but seems not to be a prognostic factor in non-endometrioid carcinomas.Objective The aim was to study the association of L1 cell adhesion molecule (L1CAM) expression with the outcome of patients with endometrial cancer, especially with regard to conventional risk variables, and to compare the patterns of relapse in L1CAM-positive and -negative cancers. Methods This was a retrospective study of 805 women. The Kaplan–Meier method and univariate and multivariate Cox regression models were applied for survival analyses. Missing data were replaced using multiple imputation. The median follow-up time was 51 months (range, 1–98). Results One hundred twenty-one (15.0%) cases were L1CAM positive. L1CAM positivity was associated with high stage (I vs II–IV) (odds ratio [OR], 2.3), lymph node involvement (OR, 2.9), poor differentiation (OR, 6.1), non-endometrioid histology (OR, 9.9), lymphovascular space invasion (OR, 2.8), cervical stromal invasion (OR, 1.8), positive peritoneal cytology (OR, 4.1), and age older than 65 years (OR, 2.8). The frequencies of deep myometrial invasion (50% or deeper), tumor size 2 cm or greater, and body mass index 30 kg/m2 or greater were not significantly different between L1CAM-positive and -negative cases. L1CAM predicted poor disease-specific survival in endometrioid (P < 0.0001) but not in non-endometrioid carcinomas (P = 0.934). The negative impact of L1CAM on outcome was confirmed in a Cox multivariate disease-specific survival analysis. Univariate survival analyses in the different ESMO–ESGO–ESTRO endometrial cancer risk groups showed an association between L1CAM positivity and poor outcome in intermediate (hazard ratio, 12) and high-risk advanced metastatic (hazard ratio, 2.0) groups. Extra-abdominal relapses were more frequent in L1CAM-positive (13.2%) than L1CAM-negative (1.9%) stage I endometrioid carcinomas (P < 0.0001), whereas other site-specific relapses in local cancers were L1CAM independent. Conclusions L1CAM is associated with the occurrence of poor prognostic variables and predicts advanced disease in endometrial cancer. L1CAM predicts extra-abdominal relapses and poor survival in endometrioid endometrial cancer, but seems not to be a prognostic factor in non-endometrioid carcinomas.

Prediction of lymphatic dissemination in endometrioid endometrial cancer: Comparison of three risk-stratification models in a single-institution cohort

OBJECTIVES To compare the performance characteristics of 3 risk-stratification models, referred to as Mayo, Helsinki and Milwaukee models, in predicting lymphatic dissemination in endometrial cancer. METHODS A total of 1052 patients with stage I-III endometrioid endometrial cancer were included in the study. The areas under curve were compared with the receiver operating characteristic curve area comparison test. Chi-square and Fisher exact test were used for comparing categorical variables. The Kaplan-Meier method and multivariable Cox regression models were used for survival analyses. The median follow-up time was 55months (range 1-108). RESULTS Areas under curve were 0.781, 0.830 and 0.829 for the Mayo, Helsinki (P=0.285 vs. Mayo) and Milwaukee (P=0.292 vs. Mayo) models, respectively, in predicting lymphatic dissemination. The rates of false negatives and false positives were similar for all models. The lymphadenectomy rate decreased in the order of Mayo model (71.5%)>Helsinki model (62.4%)>Milwaukee model (48.8%). In patients with stage I cancer, disease specific survival was better for those who satisfied low-risk criteria according to any of the models. In patients with stage II-III cancer, this difference in survival was significant only for the Milwaukee model. Both lymphatic dissemination and high-risk tumor features as per the risk models were independent predictors of survival. CONCLUSIONS The studied models had a similar accuracy in predicting lymphatic dissemination in endometrial cancer. Lymphadenectomy rate was lowest for the Milwaukee model. Survival analyses suggest that variables included in the models predict patient outcome independently of tumor stage.

L1CAM expression associates with poor outcome in endometrioid, but not in clear cell ovarian carcinoma

OBJECTIVE Our aim was to study the expression of L1CAM in endometrioid and clear cell ovarian carcinomas and to evaluate its correlation with clinical parameters and patient prognosis. METHODS Tissue microarray -based immunohistochemical analysis of L1CAM expression was performed in 249 endometrioid and 140 clear cell ovarian carcinomas. Concurrent endometrial carcinoma was found in 57 of these patients. RESULTS L1CAM expression was found in 15% of endometrioid and 23% of clear cell ovarian carcinomas. L1CAM expression was strongly associated with poor disease-specific overall survival and poor disease-free survival in endometrioid (p<0.0001, p=0.0005), but not in clear cell ovarian carcinomas. Significant association of L1CAM expression with poor overall survival was observed in grade 1-2 carcinomas (p<0.0001), but not in grade 3 tumors. In endometrioid ovarian carcinomas, L1CAM expression was associated with aggressive tumor characteristics, such as higher grade and stage, and incomplete response to primary therapy. However, L1CAM expression was not an independent prognostic factor for overall or disease-free survival. Of the 57 patients with concurrent endometrial carcinoma L1CAM positivity was found in 4 cases both in the ovarian and endometrial tumors, and in 3 cases only in the endometrial tumor. All these seven patients with L1CAM positive tumors had poor outcome. CONCLUSIONS L1CAM expression could serve as a biomarker for predicting clinical outcome and response to therapy in patients with endometrioid ovarian carcinoma, but not in clear cell carcinomas. L1CAM positivity also predicts poor outcome in patients with concurrent endometrioid ovarian and endometrial carcinomas.

Molecular changes preceding endometrial and ovarian cancer: a study of consecutive endometrial specimens from Lynch syndrome surveillance

Molecular alterations preceding endometrial and ovarian cancer and the sequence of events are unknown. Consecutive specimens from lifelong surveillance for Lynch syndrome provides a natural setting to address such questions. To molecularly define the multistep gynecological tumorigenesis, DNA mismatch repair gene mutation carriers with endometrial or ovarian carcinoma or endometrial hyperplasia were identified from a nation-wide registry and endometrial biopsy specimens taken from these individuals during 20 years of screening were collected. A total of 213 endometrial and ovarian specimens from Lynch syndrome individuals and 197 histology-matched (non-serous) samples from sporadic cases were available for this investigation. The specimens were profiled for markers linked to endometrial and ovarian tumorigenesis, including ARID1A protein expression, mismatch repair status, and tumor suppressor gene promoter methylation. In Lynch syndrome-associated endometrial and ovarian carcinomas, ARID1A protein was lost in 61–100% and mismatch repair was deficient in 97–100%, compared to 0–17% and 14–44% in sporadic cases (P = 0.000). ARID1A loss appeared in complex hyperplasia and deficient mismatch repair and tumor suppressor gene promoter methylation in histologically normal endometrium. Despite quantitative differences between Lynch syndrome and sporadic cases, ARID1A expression, mismatch repair, and tumor suppressor gene promoter methylation divided endometrial samples from both patient groups into three categories of increasing abnormality, comprising normal endometrium and simple hyperplasia (I), complex hyperplasia with or without atypia (II), and endometrial cancer (III). Complex hyperplasias without vs. with atypia were molecularly indistinguishable. In conclusion, surveillance specimens from Lynch syndrome identify mismatch repair deficiency, tumor suppressor gene promoter methylation, and ARID1A loss as early changes in tumor development. Our findings are clinically relevant for the classification of endometrial hyperplasias and have potential implications in cancer prevention in Lynch syndrome and beyond.

Prediction of Site-Specific Tumor Relapses in Patients With Stage I–II Endometrioid Endometrial Cancer

Objective The aim of this study was to investigate the association of predictors of an advanced disease and/or poor outcome with the occurrence of tumor relapses in different anatomical sites in patients with stage I–II endometrioid endometrial cancer. Methods A total of 929 patients were included in the study. The median follow-up time was 57 months (range, 1–108 months). The studied variables were: poor tumor differentiation, myometrial invasion 50% or greater, tumor size 3 cm or greater, lymphovascular space invasion, cervical stromal invasion, positive peritoneal cytology, old age (>77 years), obesity (body mass index ≥30 kg/m2), and diabetes. Results A relapse was diagnosed in 98 patients (10.5%) (vaginal in 15, pelvic in 27, intra-abdominal beyond the pelvis in 27, extra-abdominal in 29). None of the variables were associated with an altered risk of vaginal or pelvic relapses in univariate analyses. Poor differentiation, myometrial invasion 50% or greater, tumor size 3 cm or greater, and positive peritoneal cytology were associated with an increased risk of intra-abdominal relapses beyond the pelvis (odds ratios [ORs] between 2.2 and 9.6). With the exception of obesity and diabetes, all variables were associated with an increased risk of extra-abdominal relapses (ORs between 2.3 and 13). Tumor size 3 cm or greater (OR, 3.1) and positive peritoneal cytology (OR, 16) predicted intra-abdominal relapses beyond the pelvis in multivariate analysis, whereas poor differentiation (OR, 2.9), myometrial invasion 50% or greater (OR, 4.0), and positive peritoneal cytology (OR, 27) predicted extra-abdominal relapses. Compared with vaginal relapses, intra-abdominal relapses beyond the pelvis and extra-abdominal relapses were associated with a worse disease-specific survival. Survival of patients with a pelvic relapse did not differ from that of patients with a vaginal relapse. Conclusions Risk variables of endometrial cancer are differently associated with relapses in different locations. Our findings may promote studies that explore the most efficient adjuvant therapy in high-risk early-stage endometrioid endometrial cancer.

Global metabolomic profiling of uterine leiomyomas

Background:Uterine leiomyomas can be classified into molecularly distinct subtypes according to their genetic triggers: MED12 mutations, HMGA2 upregulation, or inactivation of FH. The aim of this study was to identify metabolites and metabolic pathways that are dysregulated in different subtypes of leiomyomas.Methods:We performed global metabolomic profiling of 25 uterine leiomyomas and 17 corresponding myometrium specimens using liquid chromatography–tandem mass spectroscopy.Results:A total of 641 metabolites were detected. All leiomyomas displayed reduced homocarnosine and haeme metabolite levels. We identified a clearly distinct metabolomic profile for leiomyomas of the FH subtype, characterised by metabolic alterations in the tricarboxylic acid cycle and pentose phosphate pathways, and increased levels of multiple lipids and amino acids. Several metabolites were uniquely elevated in leiomyomas of the FH subtype, including N6-succinyladenosine and argininosuccinate, serving as potential biomarkers for FH deficiency. In contrast, leiomyomas of the MED12 subtype displayed reduced levels of vitamin A, multiple membrane lipids and amino acids, and dysregulation of vitamin C metabolism, a finding which was also compatible with gene expression data.Conclusions:The study reveals the metabolomic heterogeneity of leiomyomas and provides the requisite framework for strategies designed to target metabolic alterations promoting the growth of these prevalent tumours.

Incidence of and risk factors for surgical site infections in women undergoing hysterectomy for endometrial carcinoma

The purpose of this study was to determine the incidence of, and risk factors for, surgical site infections in a contemporary cohort of women with endometrial carcinoma.