Cytokeratin 5/6 expression, prognosis, and association with estrogen receptor α in high-grade serous ovarian carcinoma.

High-grade serous ovarian carcinoma remains one of the most lethal malignancies in women. For histopathologic differentiation from mesothelioma cytokeratin, 5/6 immunohistochemistry is widely used. Another preferred marker for differential diagnosis to mesothelioma is estrogen receptor α (ER-α). In this study, we determined the rate of cytokeratin 5/6-positive cells in primary high-grade serous carcinoma. A cohort of 215 patients with high-grade serous ovarian carcinoma was evaluated immunohistochemically for the protein expression of cytokeratin 5/6. Most tumors demonstrated at least partly positive for cytokeratin 5/6 (n=148; 68.3%), showing different staining patterns from scattered stained cells to a diffuse staining, at times with a distinctive tumor-stroma border motif. Sixty-seven (31%) were entirely negative. No correlation of cytokeratin immunoreactivity score (IRS) with conventional staging parameters could be demonstrated. From the different IRS values for cytokeratin 5/6, IRS=12 (n=6; 2.9%) seemed to indicate a worse prognosis, albeit not statistically significant. An association with ER-α expression could not be detected but the combination of cytokeratin 5/6 IRS=12 and ER-α negativity resulted in a significant negative prognostic marker (overall survival: P=.003 and progression-free survival: P<.0001). We substantiate cytokeratin 5/6 protein expression as a frequent feature of high-grade serous ovarian carcinoma with various staining patterns, an important fact for the routine differential diagnosis with mesothelioma. Furthermore, cytokeratin 5/6 in combination with ER-α proved to be a negative prognostic marker, wherefore we suggest further investigation of its biological significance and possible manifestation of a basal differentiation.

Prognostic impact of HER3 based on protein and mRNA expression in high-grade serous ovarian carcinoma.

HER3 is a member of the epidermal growth factor family and was predominantly described as a negative prognostic factor in various solid tumors as well as in ovarian cancer. In this study, we investigated HER3 on protein and mRNA expression in histologically defined subtypes of ovarian cancer looking for an influence on patient's survival. Altogether, we examined HER3 in ovarian high-grade serous (HGSC, n = 320), low-grade serous (LGSC, n = 55), endometrioid (EC, n = 33), and clear cell (CCC, n = 48) carcinomas using immunohistochemistry (IHC) and quantitative real-time reverse transcription PCR (qRT-PCR). Univariate and multivariate analyses were performed to explore the association between HER3 and overall survival (OS) as well as progression-free survival (PFS). In HGSC, high HER3 mRNA expression was a favorable prognostic factor for PFS (P = 0.008) and OS (P = 0.052), while for high HER3 protein expression, a trend towards better survival was seen (OS P = 0.064; PFS P = 0.099). A subgroup of HGSC with negative HER3 staining and negative HER3 mRNA levels showed most unfavorable OS and PFS (P = 0.002 and P = 0.004, respectively). Using the multivariate Cox regression model, HER3 was predictive for prolonged PFS (HR, 0.48; 95% CI, 0.26-0.88; P = 0.018). All in all, we cannot confirm the reported negative prognostic impact of HER3 expression in high-grade serous ovarian carcinoma and moreover find a rather positive prognostic implication of HER3 in this major ovarian cancer histological subtype.

Hepatitis E virus and the safety of plasma products: investigations into the reduction capacity of manufacturing processes.

BACKGROUND: Hepatitis E virus (HEV) has been transmitted by transfusion of labile blood products and the occasional detection of HEV RNA in plasma pools indicates that HEV viremic donations might enter the manufacturing process of plasma products. To verify the safety margins of plasma products with respect to HEV, virus reduction steps commonly used in their manufacturing processes were investigated for their effectiveness to reduce HEV. STUDY DESIGN AND METHODS: Detection methods for HEV removal (by reverse transcription quantitative polymerase chain reaction) and inactivation (using an infectivity assay) were established. Immunoaffinity chromatography and 20-nm virus filtration for Factor (F)VIII, cold ethanol fractionation, and low-pH treatment for immunoglobulin, heat treatment for human albumin, and 35-nm nanofiltration for FVIII inhibitor-bypassing activity (FEIBA) were investigated for their capacity to reduce HEV or the physicochemically similar viruses feline calicivirus (FCV) and hepatitis A virus (HAV). RESULTS: For FVIII, HEV reduction of 3.9 and more than 3.9 log was demonstrated for immunoaffinity chromatography and 20-nm nanofiltration, respectively, and the cold ethanol fractionation for immunoglobulin removed more than 3.5 log of HEV, to below the limit of detection (LOD). Heat treatment of human albumin inactivated more than 3.1 log of HEV to below the LOD and 35-nm nanofiltration removed 4.0 log of HEV from the FEIBA intermediate. The results indicated HAV rather than FCV as the more relevant model virus for HEV. CONCLUSION: Substantial HEV reduction during processes commonly used in the manufacturing of plasma products was demonstrated, similar to that previously demonstrated for HAV.

Virus inactivation during the freeze-drying processes as used for the manufacture of plasma-derived medicinal products.

BACKGROUND: Freeze-drying is a technology widely used during the production of plasma-derived medicinal products. Several studies have shown that freeze-drying can also result in virus inactivation and particularly of hepatitis A virus (HAV). To date, however, the variables critical for virus inactivation during freeze-drying have not been investigated systematically. STUDY DESIGN AND METHODS: Five different lyophilization processes covering the range used for different plasma-derived medicinal products (Factor [F]VII, FVIII, F IX, FVIII inhibitor bypassing activity, and fibrin sealer protein [FSP]) were investigated for their potential to inactivate HAV as well as bovine viral diarrhea virus (BVDV) and pseudorabies virus (PRV). RESULTS: Our investigation demonstrates that freeze-drying results in significant inactivation of HAV, with reduction factors between 2.5 and 5.9 log [TCID(50)]. Also, BVDV and PRV were inactivated, although to a lesser extent. While the specific details of the freeze-drying processes investigated only had a minor influence on virus inactivation, the different compositions of product intermediates had a rather pronounced impact. CONCLUSION: Lyophilization contributes to the safety of plasma derivatives, in particular with the inactivation of HAV. The extent of HAV inactivation is strongly influenced by the respective product matrix rather than the design of the lyophilization cycle, which will require a case-to-case assessment for each product intermediate.

Inactivation of parvovirus B19 during STIM-4 vapor heat treatment of three coagulation factor concentrates.

BACKGROUND: To enhance the viral safety margins, nanofiltration has been widely integrated into the manufacturing process of plasma-derived medicinal products. Removal of smaller agents such as parvovirus B19 (B19V) by filtration, however, is typically less efficient. Because recent investigations have demonstrated that B19V may be more heat sensitive than animal parvoviruses, the potential B19V inactivation by a proprietary vapor heating procedure (STIM-4) as incorporated into the manufacturing processes of several nanofiltered coagulation factor concentrates was investigated. STUDY DESIGN AND METHODS: An infectivity assay based on quantitative reverse transcription-polymerase chain reaction (TaqMan, Applied Biosystems) detection of B19V mRNA after inoculation of a permissive cell line (UT7 Epo S1 cells) was used to investigate the virus inactivation capacity of the STIM-4 vapor heat treatment as used during the manufacture of nanofiltered second-generation Factor VIII inhibitor-bypassing activity (FEIBA), F IX complex, and FVII products. RESULTS: In contrast to animal parvoviruses, both B19V genotypes investigated, that is, 1 and 2, were shown to be surprisingly effectively inactivated by the STIM-4 vapor heat treatment process, with mean log reduction factors of 3.5 to 4.8, irrespective of the product intermediate tested. CONCLUSION: The newly demonstrated effective inactivation of B19V by vapor heating, in contrast to the earlier used animal parvoviruses, results in significant B19V safety margins for STIM-4-treated coagulation factor concentrates.

Recombinant viral sialate-O-acetylesterases.

Viral O-acetylesterases were first identified in several viruses, including influenza C viruses and coronaviruses. These enzymes are capable of removing cellular receptors from the surface of target cells. Hence they are also known as "receptor destroying" enzymes. We have cloned and expressed several recombinant viral O-acetylesterases. These enzymes were secreted from Sf9 insect cells as chimeric proteins fused to eGFP. A purification scheme to isolate the recombinant O-acetylesterase of influenza C virus was developed. The recombinant enzymes derived from influenza C viruses specifically hydrolyze 9-O-acetylated sialic acids, while that of sialodacryoadenitis virus, a rat coronavirus related to mouse hepatitis virus, is specific for 4-O-acetylated sialic acid. The recombinant esterases were shown to specifically de-O-acetylate sialic acids on glycoconjugates. We have also expressed esterase knockout proteins of the influenza C virus hemagglutinin-esterase. The recombinant viral proteins can be used to unambiguously identify O-acetylated acids in a variety of assays.

Identification and characterization of adsorbed serum sialoglycans on Leishmania donovani promastigotes.

Sialic acids as terminal residues of oligosaccharide chains play a crucial role in several cellular recognition events. The presence of sialic acid on promastigotes of Leishmania donovani, the causative organism of Indian visceral leishmaniasis, was demonstrated by fluorimetric high-performance liquid chromatography showing Neu5Ac and, to a minor extent, Neu5,9Ac2. The presence of Neu5Ac was confirmed by GC/MS analysis. Furthermore, binding with sialic acid-binding lectins Sambucus nigra agglutinin (SNA), Maackia amurensis agglutinin (MAA), and Siglecs showed the presence of both alpha2,3- and alpha2,6-linked sialic acids. No endogenous biosynthetic machinery for Neu5Ac could be demonstrated in the parasite. Concomitant western blotting of parasite membranes and culture medium with SNA demonstrated the presence of common sialoglyconjugates (123, 90, and 70 kDa). Similarly, binding of MAA with parasite membrane and culture medium showed three analogous sialoglycans corresponding to 130, 117, and 70 kDa, indicating that alpha2,3- and alpha2,6-linked sialoglycans are adsorbed from the fetal calf serum present in the culture medium. L. donovani promastigotes also reacted with Achatinin-H, a lectin that preferentially identifies 9-O-acetylated sialic acid in alpha2-->6 GalNAc linkage. This determinant was evidenced on parasite cell surfaces by cell agglutination, ELISA, and flow cytometry, where its binding was abolished by pretreatment of cells with a recombinant 9-O-acetylesterase derived from the HE1 region of the influenza C esterase gene. Additionally, binding of CD60b, a 9-O-acetyl GD3-specific monoclonal antibody, corroborated the presence of terminal 9-O-acetylated disialoglycans. Our results indicate that sialic acids (alpha2-->6 and alpha2-->3 linked) and 9-O-acetyl derivatives constitute components of the parasite cell surface.