Katja Vouk

Discovery of phosphatidylcholines and sphingomyelins as biomarkers for ovarian endometriosis

BACKGROUND Current non-invasive diagnostic methods for endometriosis lack sensitivity and specificity. In search for new diagnostic biomarkers for ovarian endometriosis, we used a hypothesis-generating targeted metabolomics approach. METHODS In a case-control study, we collected plasma of study participants and analysed their metabolic profiles. We selected a group of 40 patients with ovarian endometriosis who underwent laparoscopic surgery and a control group of 52 healthy women who underwent sterilization at the University Clinical Centre Ljubljana, Slovenia. Over 140 targeted analytes included glycerophospholipids, sphingolipids and acylcarnitines. The analytes were quantified by electrospray ionization tandem mass spectrometry. For assessing the strength of association between the metabolite or metabolite ratios and the disease, we used crude and adjusted odds ratios. A stepwise logistic regression procedure was used for selecting the best combination of biomarkers. RESULTS Eight lipid metabolites were identified as endometriosis-associated biomarkers due to elevated levels in patients compared with controls. A model containing hydroxysphingomyelin SMOH C16:1 and the ratio between phosphatidylcholine PCaa C36:2 to ether-phospholipid PCae C34:2, adjusted for the effect of age and the BMI, resulted in a sensitivity of 90.0%, a specificity of 84.3% and a ratio of the positive likelihood ratio to the negative likelihood ratio of 48.3. CONCLUSIONS Our results suggest that endometriosis is associated with elevated levels of sphingomyelins and phosphatidylcholines, which might contribute to the suppression of apoptosis and affect lipid-associated signalling pathways. Our findings suggest novel potential routes for therapy by specifically blocking highly up-regulated isoforms of phosphpolipase A2 and lysophosphatidylcholine acyltransferase 4.

Aldo-keto reductases AKR1C1, AKR1C2 and AKR1C3 may enhance progesterone metabolism in ovarian endometriosis.

Endometriosis is a very common disease that is characterized by increased formation of estradiol and disturbed progesterone action. This latter is usually explained by a lack of progesterone receptor B (PR-B) expression, while the role of pre-receptor metabolism of progesterone is not yet fully understood. In normal endometrium, progesterone is metabolized by reductive 20α-hydroxysteroid dehydrogenases (20α-HSDs), 3α/β-HSDs and 5α/β-reductases. The aldo-keto reductases 1C1 and 1C3 (AKR1C1 and AKR1C3) are the major reductive 20α-HSDs, while the oxidative reaction is catalyzed by 17β-HSD type 2 (HSD17B2). Also, 3α-HSD and 3β-HSD activities have been associated with the AKR1C isozymes. Additionally, 5α-reductase types 1 and 2 (SRD5A1, SRD5A2) and 5β-reductase (AKR1D1) are responsible for the formation of 5α- and 5β-reduced pregnanes. In this study, we examined the expression of PR-AB and the progesterone metabolizing enzymes in 31 specimens of ovarian endometriosis and 28 specimens of normal endometrium. Real-time PCR analysis revealed significantly decreased mRNA levels of PR-AB, HSD17B2 and SRD5A2, significantly increased mRNA levels of AKR1C1, AKR1C2, AKR1C3 and SRD5A1, and negligible mRNA levels of AKR1D1. Immunohistochemistry staining of endometriotic tissue compared to control endometrium showed significantly lower PR-B levels in epithelial cells and no significant differences in stromal cells, there were no significant differences in the expression of AKR1C3 and significantly higher AKR1C2 levels were seen only in stromal cells. Our expression analysis data at the mRNA level and partially at the cellular level thus suggest enhanced metabolism of progesterone by SRD5A1 and the 20α-HSD and 3α/β-HSD activities of AKR1C1, AKR1C2 and AKR1C3.

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

Abstract 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.

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.

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.

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

Abstract 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.

K5 D328E: A Novel Missense Mutation in the Linker 12 Domain of Keratin 5 Associated with Epidermolysis bullosa simplex (Weber-Cockayne)

A novel missense mutation was detected in the L12 region of keratin 5 (K5) in a Slovene family diagnosed with a Weber-Cockayne variant of epidermolysis bullosa simplex (EBS). Direct sequencing identified a heterozygous GAC to GAA substitution altering codon 328 of K5 from Asp to Glu in all affected family members, while no mutation was observed either in the healthy individual or the 50 unrelated control samples. Asp328 of K5 (position 12 in the L12 domain) is remarkably conserved among all type II keratins. K5 L12:D12E is the third mutation found to affect this residue in K5-related EBS, indicating the importance of Asp328 for K5 structure and the dramatic effect that fine changes can have on keratin intermediate filament integrity.

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

BackgroundAutosomal 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.MethodsWe 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 .ResultsLod 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.ConclusionIn 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.

Screen of 55 Slovenian haemophilia A patients: Identification of 2 novel mutations (S‐1R and IVS23+1G→A) and discussion of mutation spectrum

Using polymerase chain reaction, single‐stranded conformational polymorphism (SSCP), TaqI restriction analysis and direct sequencing, exons 1, 7, 8, 9, 12, 13, 14, 18, 22, 23, 24, and 26 of the factor VIII gene were screened for point mutations in 55 Slovenian haemophilia A patients. In eighteen patients eleven different mutations were found; one (in six patients) in exon 26, one (in two patients) in exon 24, two in exon 23, one in intron 23, one in exon 18, one in exon 12, one in exon 8, two (1 + 1 in two patients) in exon 7 and one in exon 1. Of the mutations detected one has recently been reported by us (Q602X), and two are novel; S‐1R in exon 1 and IVS23+1G→A in intron 23. Hum Mutat 13:413–413, 1999.

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.