Next-generation sequencing identifies novel mitochondrial variants in pituitary adenomas.

PURPOSE: Disrupted mitochondrial functions and genetic variants of mitochondrial DNA (mtDNA) have been observed in different human neoplasms. Next-generation sequencing (NGS) can be used to detect even low heteroplasmy-level mtDNA variants. We aimed to investigate the mitochondrial genome in pituitary adenomas by NGS. METHODS: We analysed 11 growth hormone producing and 33 non-functioning [22 gonadotroph and 11 hormone immunonegative] pituitary adenomas using VariantPro™ Mitochondrion Panel on Illumina MiSeq instrument. Revised Cambridge Reference Sequence (rCRS) of the mtDNA was used as reference. Heteroplasmy was determined using a 3% cutoff. RESULTS: 496 variants were identified in pituitary adenomas with overall low level of heteroplasmy (7.22%). On average, 35 variants were detected per sample. Samples harbouring the highest number of variants had the highest Ki-67 indices independently of histological subtypes. We identified eight variants (A11251G, T4216C, T16126C, C15452A, T14798C, A188G, G185A, and T16093C) with different prevalences among different histological groups. T16189C was found in 40% of non-recurrent adenomas, while it was not present in the recurrent ones. T14798C and T4216C were confirmed by Sanger sequencing in all 44 samples. 100% concordance was found between NGS and Sanger method. CONCLUSIONS: NGS is a reliable method for investigating mitochondrial genome and heteroplasmy in pituitary adenomas. Out of the 496 detected variants, 414 have not been previously reported in pituitary adenoma. The high number of mtDNA variants may contribute to adenoma genesis, and some variants (i.e., T16189C) might associate with benign behaviour.

Circulating miRNAs as biomarkers for endocrine disorders.

Specific, sensitive and non-invasive biomarkers are always needed in endocrine disorders. miRNAs are short, non-coding RNA molecules with well-known role in gene expression regulation. They are frequently dysregulated in metabolic and endocrine diseases. Recently it has been shown that they are secreted into biofluids by nearly all kind of cell types. As they can be taken up by other cells they may have a role in a new kind of paracrine, cell-to-cell communication. Circulating miRNAs are protected by RNA-binding proteins or microvesicles hence they can be attractive candidates as diagnostic or prognostic biomarkers. In this review, we summarize the characteristics of extracellular miRNA's and our knowledge about their origin and potential roles in endocrine and metabolic diseases. Discussions about the technical challenges occurring during identification and measurement of extracellular miRNAs and future perspectives about their roles are also highlighted.

Preoperative HE4 expression in plasma predicts surgical outcome in primary ovarian cancer patients: results from the OVCAD study.

OBJECTIVES: Epithelial ovarian cancer (EOC) is the major cause of death due to gynecological malignancies. The most important prognostic factors are residual tumor mass after surgery and platinum-response. No predictive biomarkers are available to identify patients who will benefit from standard treatment. The aim of our study was to analyze the role of HE4 in predicting surgical and clinical outcome in primary EOC. METHODS: In the European multicentric project "OVCAD", 275 consecutive patients with primary EOC were enrolled. Patients were eligible if radical cytoreductive surgery was performed and platinum-based chemotherapy was applied. Plasma and ascites samples were collected before or during surgery. The concentrations of HE4 and CA125 was determined using ELISA and Luminex technique, respectively. RESULTS: Median age at first diagnosis was 58 years (range 18-85 years). Most patients presented with advanced stage disease, FIGO III or IV (94.6%), grades II-III (96%) and serous histology (86.2%). In most cases a complete cytoreduction to no residual tumor mass was achieved (68.4%). Higher plasma HE4 levels correlated with poor surgery outcome in terms of macroscopically residual tumor mass (p<0.001) and platinum-resistance (p=0.009). Plasma CA125 and the risk index (HE4 and CA125) were independent predictive factors for surgical outcome (p=0.001, OR=3.37, 95% CI=1.61-7.06 and p<0.001, OR=6,041, 95% CI=2.33-15.65, respectively). FIGO stage III was an independent predictive factor for platinum response (p=0.039, OR=0.436, 95% CI=0.198-0.960). CONCLUSIONS: The presented data are showing that the combination of HE4 and CA125 expression in plasma might predict the surgical outcome in EOC and by this may have a prognostic impact on PFS and OS.

Secreted and cellular proteochondroitin sulfates of a human B lymphoblastoid cell line contain different protein cores.

Proteoglycans of the human B lymphoblastoid cell line LICR-LON-HMy2 were metabolically labeled with [35S]sulfate. High-density fractions of 35S-labeled material separated by CsCl gradient ultracentrifugation were further purified by anion exchange chromatography and gel filtration. Two proteoglycans, isolated from cell lysates and culture supernatants, were characterized by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in combination with enzymatic degradation. Treatment with chondroitinase AC completely degraded the glycosaminoglycan moiety of the proteoglycans. Three to 4 chondroitin sulfate chains (average molecular mass = 26 kDa) were estimated for each of the two proteoglycans. Differences between the proteochondroitin sulfates (CSPG) were observed in the content of N-linked oligosaccharides. After chondroitinase AC treatment the resulting band in SDS-PAGE of the secreted CSPG was sensitive to treatment with endoglycosidase F (Endo F) which further reduced the molecular mass from 30 to 21.5 kDa, whereas the band of the cellular CSPG after chondroitinase AC treatment (molecular mass = 30 kDa) remained resistant to Endo F treatment. The composition of amino acids was different in the protein cores, suggesting differences in the primary structure. Both CSPG contained a high percentage of glycine and serine. For both CSPG a molecular mass of approximately 135 kDa was deduced from the hydrodynamic sizes of the glycosaminoglycan chains obtained after alkaline/borohydride treatment and the migration of the protein/oligosaccharide complexes in SDS-PAGE. 75% of all [35S]sulfate-labeled molecules were found in the culture supernatant and 25% in the cellular fraction. 35S-Labeled material in the culture supernatant consisted exclusively of intact CSPG, whereas 35S-Labeled molecules in the cellular preparation consisted largely of free chondroitin sulfate chains. Only 8.3% of the cellular material, isolated from the microsomal fraction, was intact CSPG. In pulse-chase experiments maximal secretion of CSPG was found after 4 h, comprising approximately 40% of totally synthesized CSPG. From these experiments we tentatively conclude that a small proportion of CSPG synthesized by LICR-LON-HMy2 cells is membrane-associated, a larger portion is secreted, and another portion is intracellularly degraded.

Development of a luminescence immunoassay for follitropin suitable for clinical routine.

We developed a luminescence immunoassay (LIA) for follitropin, based on the synthesis of a follitropin-N-(4-aminobutyl)-N-ethylisoluminol conjugate. The luminescence tracer was purified by gel chromatography. Antibody-bound and non-bound tracer fractions were separated by using a second antibody reagent bound to magnetic particles. The assay can be performed within 24 hours and is sufficiently sensitive for the measurement of all clinically relevant follitropin concentrations including the subnormal range.