Novel algorithm including CA-125, HE4 and body mass index in the diagnosis of endometrial cancer.

OBJECTIVES: To evaluate the diagnostic and prognostic potential of preoperative serum CA-125 and HE4 levels in patients with endometrial cancer. METHODS: Prospective case-control study of 133 women who underwent surgical treatment at the University Medical Centre Ljubljana (64 patients with endometrial cancer, 69 control patients with prolapsed uterus or myoma). Serum CA-125 and HE4 levels were determined using electrochemiluminescent assays. RESULTS: Serum CA-125 and HE4 levels were significantly higher in patients with endometrial cancer, compared to the controls (p=2.67×10-4, 1.36×10-7, respectively). A diagnostic model that combines serum CA-125 and HE4 levels and body mass index separated patients with endometrial cancer from controls, with AUC of 0.804, sensitivity of 66.7%, and specificity of 84.6%. Serum HE4 levels showed good prognostic potential and stratified the patients according to presence/absence of deep myometrial invasion (p=0.001) or lymphovascular invasion (p=0.003), with AUCs of 0.78 and 0.81, respectively. In low-risk patients with grade 1 and 2 endometrioid cancer for whom lymphadenectomy can be avoided, HE4 allowed stratification according to deep myometrial invasion (p=3.39×10-4), with AUC of 0.84. Although median HE4 levels were higher in patients with lymphovascular invasion, this difference did not reach significance (p=0.06). CONCLUSIONS: A model based on preoperative serum CA-125 and HE4 levels and body mass index has good diagnostic accuracy for separation of patients with endometrial cancer and control patients. In patients with endometrial cancer, serum HE4 levels allow prediction of deep myometrial and lymphovascular invasion.

Elevated glycodelin-A concentrations in serum and peritoneal fluid of women with ovarian endometriosis.

The aim of this study was to evaluate serum and peritoneal fluid (PF) glycodelin-A concentrations in women with ovarian endometriosis. Ninety-nine matched pairs of serum and PF samples were included in our study. The case group comprised 57 women with ovarian endometriosis and the control group 42 healthy women undergoing sterilization or patients with benign ovarian cysts. Glycodelin-A concentrations were measured using ELISA. Endometriosis patients had significantly higher serum and PF glycodelin-A concentrations compared to controls, and this increase was observed in both proliferative and secretory cycle phases. Glycodelin-A concentrations were more than 10-fold higher in PF than in serum and correlated with each other. Intensity and frequency of menstrual pain positively correlated with glycodelin-A concentrations. Sensitivity and specificity of glycodelin-A as a biomarker for ovarian endometriosis were 82.1% and 78.4% in serum, and 79.7% and 77.5% in PF, respectively. These results indicate that Glycodelin-A has a potential role as a biomarker to be used in combination with other, independent marker molecules.

Models including plasma levels of sphingomyelins and phosphatidylcholines as diagnostic and prognostic biomarkers of endometrial cancer.

In endometrial cancer, biomarkers for preoperative identification of patients with low risk for disease progression would enable stratification according to the extent of surgery needed, and would avoid the complications that can be associated with radical surgery. A panel of proteins, amino acids, enzymes, and miRNA has been investigated as potential biomarkers for endometrial cancer. At the time of the manuscript submission targeted metabolomics/lipidomics approaches have not been applied to biomarker research in endometrial cancer. Using electrospray ionization-tandem mass spectrometry we quantified 163 metabolites in 126 plasma samples (61 patients with endometrial cancer, 65 control patients). Three single phosphatidylcholines were identified with significantly decreased levels in patients with endometrial cancer. A diagnostic model was defined as the ratio between acylcarnitine C16 and phosphatidylcholine PCae C40:1, the ratio between proline and tyrosine, and the ratio between the two phosphatidylcholines PCaa C42:0 and PCae C44:5; which provided sensitivity of 85.25%, specificity of 69.23%, and AUC of 0.837. Addition of smoking status further improved the constructed diagnostic model (AUC = 0.855). The presence of the major prognostic factors of deep myometrial invasion and lymphovascular invasion were also associated with altered metabolite concentrations. A prognostic model for deep myometrial invasion included the ratio between two hydroxysphingomyelins SMOH C14:1 and SMOH C24:1, and the ratio between two phosphatidylcholines PCaa C40:2 and PCaa C42:6, which provided sensitivity of 81.25%, specificity of 86.36%, and AUC of 0.857. The model for lymphovascular invasion included the ratio between two phosphatidylcholines PCaa C34:4 and PCae C38:3, and the ratio between acylcarnitine C16:2 and phosphatidylcholine PCaa C38:1, which provided sensitivity of 88.89%, specificity of 84.31%, and AUC of 0.935.

Models including serum CA-125, BMI, cyst pathology, dysmenorrhea or dyspareunia for diagnosis of endometriosis.

AIM: To evaluate preoperative levels of CA-125 and HE4 in patients with endometriosis-like symptoms and to construct diagnostic models. PATIENTS: Prospective case-control study included 124 endometriosis patients and 97 control patients. MATERIALS & METHODS: Logistic regression was used to construct diagnostic models based on serum biomarker levels and clinical data. RESULTS: A model with CA-125, BMI, information on cysts and dyspareunia had an area under the curve value of 0.836, sensitivity of 74.0% and specificity of 81.3%. The second model included CA-125, BMI, information on cysts and dysmenorrhea and had an area under the curve value of 0.819, sensitivity of 74.8% and specificity of 79.2%. CONCLUSION: Constructed models have the potential for noninvasive diagnosis of endometriosis, and might be translated into clinical practice after additional validation.

The Significance of the Sulfatase Pathway for Local Estrogen Formation in Endometrial Cancer.

Endometrial cancer (EC) is the most common estrogen-dependent gynecological malignancy in the developed World. To investigate the local formation of estradiol (E2), we first measured the concentrations of the steroid precursor androstenedione (A-dione) and the most potent estrogen, E2, and we evaluated the metabolism of A-dione, estrone-sulfate (E1-S), and estrone (E1) in cancerous and adjacent control endometrium. Furthermore, we studied expression of the key genes for estradiol formation via the aromatase and sulfatase pathways. A-dione and E2 were detected in cancerous and adjacent control endometrium. In cancerous endometrium, A-dione was metabolized to testosterone, and no E2 was formed. Both, E1-S and E1 were metabolized to E2, with increased levels of E2 seen in cancerous tissue. There was no significant difference in expression of the key genes of the aromatase (CYP19A1) and the sulfatase (STS, HSD17B1, HSD17B2) pathways in cancerous endometrium compared to adjacent control tissue. The mRNA levels of CYP19A1 and HSD17B1 were low, and HSD17B14, which promotes inactivation of E2, was significantly down-regulated in cancerous endometrium, especially in patients with lymphovascular invasion. At the protein level, there were no differences in the levels of STS and HSD17B2 between cancerous and adjacent control tissue by Western blotting, and immunohistochemistry revealed intense staining for STS and HSD17B2, and weak staining for SULT1E1 and HSD17B1 in cancerous tissue. Our data demonstrate that in cancerous endometrium, E2 is formed from E1-S via the sulfatase pathway, and not from A-dione via the aromatase pathway.

PKD1 and PKD2 mutations in Slovenian families with autosomal dominant polycystic kidney disease.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is a genetically heterogeneous disorder caused by mutations in at least two different loci. Prior to performing mutation screening, if DNA samples of sufficient number of family members are available, it is worthwhile to assign the gene involved in disease progression by the genetic linkage analysis. METHODS: We collected samples from 36 Slovene ADPKD families and performed linkage analysis in 16 of them. Linkage was assessed by the use of microsatellite polymorphic markers, four in the case of PKD1 (KG8, AC2.5, CW3 and CW2) and five for PKD2 (D4S1534, D4S2929, D4S1542, D4S1563 and D4S423). Partial PKD1 mutation screening was undertaken by analysing exons 23 and 31-46 and PKD2 . RESULTS: Lod scores indicated linkage to PKD1 in six families and to PKD2 in two families. One family was linked to none and in seven families linkage to both genes was possible. Partial PKD1 mutation screening was performed in 33 patients (including 20 patients from the families where linkage analysis could not be performed). We analysed PKD2 in 2 patients where lod scores indicated linkage to PKD2 and in 7 families where linkage to both genes was possible. We detected six mutations and eight polymorphisms in PKD1 and one mutation and three polymorphisms in PKD2. CONCLUSION: In our study group of ADPKD patients we detected seven mutations: three frameshift, one missense, two nonsense and one putative splicing mutation. Three have been described previously and 4 are novel. Three newly described framesfift mutations in PKD1 seem to be associated with more severe clinical course of ADPKD. Previously described nonsense mutation in PKD2 seems to be associated with cysts in liver and milder clinical course.

Altered levels of acylcarnitines, phosphatidylcholines, and sphingomyelins in peritoneal fluid from ovarian endometriosis patients.

Endometriosis is a complex, polygenic, and estrogen-dependent disease that affects 6% to 10% of women of reproductive age, and 30% to 50% of women with infertility and/or pelvic pain. Surgical diagnosis of endometriosis is still the gold standard, as there are currently no diagnostic biomarkers available. Due to the invasive diagnostics, it can take up to 11 years before affected women are diagnosed and receive the appropriate treatment. We performed a targeted metabolomics study to search for potential semi-invasive biomarkers in peritoneal fluid from endometriosis patients. Our case-control study comprised 29 ovarian endometriosis patients and 36 healthy control women. The 148 metabolites included acylcarnitines, glycerophospholipids, and sphingolipids, which were quantified by electrospray ionization tandem mass spectrometry. The strength of association between the metabolites and the metabolite ratios and disease was assessed using crude and adjusted odds ratios. The best combination of biomarkers was then selected by performing step-wise logistic regression. Our analysis reveals significantly decreased concentrations of 10 metabolites, of carnitine and acylcarnitines (C0, C8:1, C6C4:1 DC, C10:1), phosphatidylcholines (PC aa C38:3, PC aa C38:4, PC aa C40:4, PC aa C40:5), and sphingomyelins (SM C16:1, SM C18:1), and 125 significantly altered metabolite ratios in patients versus control women. The best model includes two ratios: a carnitine to a phosphatidylcholine (C0/PC ae C36:0); and between two phosphatidylcholines (PC aa C30:0/PC ae C32:2). When adjusted for age, this provides sensitivity of 82.8% and specificity of 94.4%, with AUC of 0.944. Our study supports the importance of carnitine, phosphatidylcholine, and sphingomyelin metabolites in the pathophysiology of endometriosis, and confirms the potential for the combination of individual metabolite ratios to provide biomarkers for semi-invasive diagnostics.

Cost and economic burden of adverse events associated with metastatic melanoma treatments in five countries.

OBJECTIVE: To estimate per-event cost and economic burden associated with managing the most common and/or severe metastatic melanoma (MM) treatment-related adverse events (AEs) in Australia, France, Germany, Italy, and the UK. METHODS: AEs associated with chemotherapy (dacarbazine, paclitaxel, fotemustine), immunotherapy (ipilimumab), and targeted therapy (vemurafenib) were identified by literature review. Medical resource use data associated with managing AEs were collected through two blinded Delphi panel cycles in each of the five countries. Published costs were used to estimate per-event costs and combined with AEs incidence, treatment usage, and MM prevalence to estimate the economic burden for each country. RESULTS: The costliest AEs were grade 3/4 events due to immunotherapy (Australia/France: colitis; UK: diarrhea) and chemotherapy (Germany/Italy: neutropenia/leukopenia). Treatment of AEs specific to chemotherapy (Australia/Germany/Italy/France: neutropenia/leukopenia) and targeted therapy (UK: squamous cell carcinoma) contributed heavily to country-specific economic burden. LIMITATIONS: Economic burden was estimated assuming that each patient experienced an AE only once. In addition, the context of settings was heterogeneous and the number of Delphi panel experts was limited. CONCLUSIONS: Management costs for MM treatment-associated AEs can be substantial. Results could be incorporated in economic models that support reimbursement dossiers. With the availability of newer treatments, establishment of a baseline measure of the economic burden of AEs will be crucial for assessing their impact on patients and regional healthcare systems.

Panels of cytokines and other secretory proteins as potential biomarkers of ovarian endometriosis.

Endometriosis is a gynecologic disease that is characterized by nonspecific symptoms and invasive diagnostics. To date, there is no adequate noninvasive method for the diagnosis of endometriosis. Although more than 100 potential biomarkers have been investigated in blood and/or peritoneal fluid, none of these has proven useful in clinical practice. The aim to find a suitable panel of biomarkers that would allow noninvasive diagnosis thus remains of interest. We evaluated the concentrations of 16 cytokines and other secretory proteins in serum and peritoneal fluid of 58 women with ovarian endometriosis (cases) and 40 healthy women undergoing sterilization or patients with benign ovarian cysts (controls) using multiplexed double fluorescence-based immunometric assay platform and enzyme-linked immunosorbent assay. Significantly higher concentrations of glycodelin-A were shown in serum, and significantly higher levels of glycodelin-A, IL-6, and IL-8, and lower levels of leptin were measured in the peritoneal fluid of cases versus controls. In serum, the best performance was shown by models that included the ratio of leptin/glycodelin-A and the ratio of ficolin 2/glycodelin-A, whereas in the peritoneal fluid the best models included the ratio of biglycan/leptin, regulated on activation normal T-cell expressed and secreted/IL-6 and ficolin-2/glycodelin-A, and IL-8 per milligram of total protein, all in combination with age. The models using serum and peritoneal fluid distinguished between ovarian endometriosis patients and controls regardless of the menstrual cycle phase with relatively high sensitivity (72.5% to 84.2%), specificity (78.4% to 91.2%), and area under the curve (0.85 to 0.90).

Mutational analysis of 30 Slovenian cystic fibrosis patients compared to known Slovenian and European CF mutation spectra.

More than 800 mutations have been indentified in the CFTR gene. This vast mutation diversity makes the search for molecular defects in cystic fibrosis difficult. Out of 100 Slovenian CF families, we have screened 30, using DGGE and SSCP as mutation detection techniques, while the remaining 70 have been studied previously. Together our and the previous studies have been able to indentify 18 CF mutations which cover 77.6% of the CF alleles in those families. The relative frequency of ΔF508 is 62.7% which is significantly higher than the average reported for the Mediterranean South European region (51.6%). At the same time, significant differences in mutation frequencies were found for the G542X, R1162X, W1282X, N1303K and 3905insT mutations. Several, otherwise rare mutations have been detected, such as: I148T, Q552X, 457TAT→G, R1006H, 2907delTT, 3667ins4, A559T and G576A. An interesting fact is that A559T was so far found mostly in CF patients of African-American origin. These results imply that a high heterogeneity of CF mutations occurs within the small population of Slovenia, consisting only of 2 million inhabitants. In view of the spectrum and frequencies of detected mutations, Slovenian population expresses characteristics of Mediterranean and central European countries, and at the same time shows also distinctive differences and unique region specific CF mutations (Q685X, D192G, S4X).

Will vaccination against rotavirus infection with RIX4414 be cost-saving in Germany?

BACKGROUND: Rotavirus gastroenteritis (RVGE) is a frequent disease in young children. The recommended German paediatric immunisation schedule does not currently include rotavirus vaccination. A lack of economic data on the impact of routine vaccination is stated as one of the reasons. As a result, the current coverage rate is low, around 26%. This study investigated whether rotavirus vaccination using the two-dose rotavirus vaccine RIX4414 (Rotarix®, GlaxoSmithKline Vaccines) would be a cost-saving intervention from the perspective of the statutory health insurance (SHI) in Germany. OBJECTIVE: The objective of the study was to analyse health outcomes (number of RVGE cases and hospitalisations prevented) and the associated cost to the SHI when comparing 100% rotavirus vaccination with no vaccination in Germany. METHODS: A Markov cohort model simulated the number of RVGE events and related costs in a German birth cohort over the first 60 months of life with current disease management. The model compared an unvaccinated cohort with a fully vaccinated cohort. Vaccine efficacy data from international clinical trials were combined with German-specific epidemiological and cost data. Results were tested using extensive sensitivity analyses. RESULTS: Full vaccination of a birth cohort against rotavirus disease would be expected to prevent 82% of RVGE cases, reducing RVGE frequency from 28 to 5 events per 100 children in the birth cohort up to age 5 years. The estimated cost reduction with vaccination for that period is predicted to be €9.2 million with 100% coverage (€6.9 million with 75% coverage), mainly due to reductions in SHI reimbursement for productivity losses, hospital stays and visits to office-based physicians due to the vaccine's efficacy against severe disease. CONCLUSIONS: Routine rotavirus vaccination in Germany would reduce the number of hospitalised and outpatient cases. The associated investment could be fully offset by costs avoided in hospital stays, physician visits and SHI reimbursement of productivity losses. Sensitivity analysis indicated that vaccination would be cost-saving in 95% of simulations. Incremental cost was observed only under extreme conditions, especially when the time spent at home due to rotavirus disease was low or when vaccine efficacy against severe disease was heavily decreased.

Novel estrogen-related genes and potential biomarkers of ovarian endometriosis identified by differential expression analysis.

In the search for novel biomarkers of endometriosis, we selected 152 genes from the GeneLogic database based on results of genome-wide expression analysis of ovarian endometriosis, plus 20 genes related to estrogen metabolism and action. We then performed low-density array analysis of these 172 genes on 11 ovarian endometriosis samples and 9 control endometrium samples. Principal component analysis of the gene expression levels showed clear separation between the endometriosis and control groups. We identified 78 genes as differentially expressed. Based on Ingenuity pathway analysis, these differentially expressed genes were arranged into groups according to biological function. These analyses revealed that 32 differentially expressed genes are estrogen related, 23 of which have not been reported previously in connection with endometriosis. Functional annotation showed that 25 and 22 genes are associated with the biological terms "secreted" and "extracellular region", respectively. Differential expression of 4 out of 5 genes related to estrogen metabolism and action (ESR1, ESR2, PGR and BGN) was also confirmed by immunohistochemistry. Our study thus reveals differential expression of several genes that have not previously been associated with endometriosis and that encode potential novel biomarkers and drug targets.