Case-mix adjustment to compare hospital performances regarding complications after cytoreductive surgery for ovarian cancer: a nationwide population-based study.

OBJECTIVE: Complication rates after cytoreductive surgery are important quality indicators for hospitals that treat patients with advanced-stage ovarian cancer. Case-mix factors are patient and tumor characteristics that may influence hospital outcomes such as the complication rates. Currently, no case-mix adjustment model exists for complications after cytoreductive surgery; therefore, it is unclear whether hospitals are being compared correctly. This study aims to develop the first case-mix adjustment model for complications after surgery for advanced-stage ovarian cancer, enabling an accurate comparison between hospitals. METHODS: This population-based study included all patients undergoing cytoreductive surgery for advanced-stage ovarian cancer registered in the Netherlands in 2017-2019. Case-mix variables were identified and assessed using logistic regressions. The primary outcome was the composite outcome measure 'complicated course'. Patients had a complicated course when at least one of the following criteria were met: (1) any complication combined with a prolonged length of hospital stay; (2) complication requiring reintervention; (3) any complication with a prolonged length of stay in the intensive care unit; or (4) 30-day mortality or in-hospital mortality during admission following surgery. Inter-hospital variation was analyzed using univariable and multivariable logistic regressions and visualized using funnel plots. RESULTS: A total of 1822 patients were included, of which 10.7% (n=195) had a complicated course. Comorbidity and tumor stage had a significant impact on complicated course rates in multivariable logistic regression. Inter-hospital variation was not significant for case-mix factors. Complicated course rates ranged between 2.2% and 29.1%, and case-mix adjusted observed/expected ratios ranged from 0.20 to 2.67 between hospitals. Three hospitals performed outside the confidence intervals for complicated course rates. These hospitals remained outliers after case-mix adjustment. CONCLUSION: There is variation between hospitals regarding complicated course rates after cytoreductive surgery for ovarian cancer in the Netherlands. While comorbidity and tumor stage significantly affected the complicated course rates, adjusting for case-mix factors did not significantly affect hospital outcomes. The limited impact of case-mix adjustment could be a result of the Dutch centralized healthcare model.

Guideline adherence in ovarian cancer for surgical staging in the Netherlands.

OBJECTIVE: Previous studies have shown low adherence to surgical staging guidelines in patients with clinical early-stage ovarian carcinoma. The aim of this study was to identify guideline adherence for surgical staging and to show the distribution of each surgical item within the study population. In addition, we examined whether regional variation in the Netherlands exists for complete surgical staging. METHODS: Patients with ovarian cancer and surgical staging registered in the Dutch Gynecological Oncology Audit between January 1, 2015 and December 31, 2019 in the Netherlands were included. Complete surgical staging was defined according to the Dutch evidence-based guideline. Surgical items were ranked and illustrated. Variation in complete surgical staging for eight regional cancer networks was shown in funnel plots. Manual validation of registered data was performed in three gynecological oncology centers. RESULTS: 604 patients underwent surgical staging, 365 (60%) underwent an incomplete staging procedure, 295 (81%) were registered with early-stage disease (International Federation of Gynecology and Obstetrics I-IIA) and, of these patients, 115 (39%) received adjuvant chemotherapy. Patients with incomplete surgical staging were operated more often with minimal invasive techniques (laparoscopy or robot) compared with patients in the complete staging group (p<0.001). Sampling of cytology/ascites was the most frequently lacking factor (29%). Manual validation of data in three gynecological oncology centers identified reasons for incomplete staging, the most common being 'perioperative findings' such as dense adhesions between tumor and peritoneum, consistent with advanced stage disease (≥IIA). Regional variation for complete surgical staging showed two regions performing outside the confidence intervals (12.5% and 25.5%, mean 40%). CONCLUSION: Guideline adherence for staging was lower than expected and validation of data gave additional insights into the reasons that were contributing to incomplete surgical staging. Moreover, this analysis showed that regional variation for surgical staging exists, which forms a starting point to improve and harmonize staging procedures for these patients nationwide.

The role of chemical speciation, chemical fractionation and calcium disruption in manganese-induced developmental toxicity in zebrafish (Danio rerio) embryos.

Manganese (Mn) is an essential nutrient that can be toxic in excess concentrations, especially during early development stages. The mechanisms of Mn toxicity is still unclear, and little information is available regarding the role of Mn speciation and fractionation in toxicology. We aimed to investigate the toxic effects of several chemical forms of Mn in embryos of Danio rerio exposed during different development stages, between 2 and 122h post fertilization. We found a stage-specific increase of lethality associated with hatching and removal of the chorion. Mn(II), ([Mn(H2O)6](2+)) appeared to be the most toxic species to embryos exposed for 48h, and Mn(II) citrate was most toxic to embryos exposed for 72 and/or 120h. Manganese toxicity was associated with calcium disruption, manganese speciation and metal fractionation, including bioaccumulation in tissue, granule fractions, organelles and denaturated proteins.

NADP+ -dependent IDH1 R132 mutation and its relevance for glioma patient survival.

The isocitrate dehydrogenase 1 (IDH1) mutation occurs in high frequency in glioma and secondary glioblastoma (GBM). Mutated IDH1 produces the oncometabolite 2-hydroxyglutarate rather than α-ketoglutarate or isocitrate. The oncometabolite is considered to be the major cause of the association between the IDH1 mutation and gliomagenesis. On the other hand, the IDH1 mutation in GBM is associated with prolonged patient survival. This association is not well understood yet but IDH1 involvement in epigenetic silencing of O-6-methylguanine-DNA methyltransferase (MGMT), a DNA repair enzyme is considered to be an important mechanism. However, it was shown recently that the IDH1 mutation and MGMT silencing are independent prognostic factors. Here, we hypothesize that the IDH1 mutation reduces the capacity to produce NADPH and thus reduces the capacity to scavenge reactive oxygen species that are generated during irradiation and chemotherapy. IDH1 activity is responsible for two-thirds of the NADPH production capacity in normal brain, whereas the IDH1 mutation reduces this capacity by almost 40%. Therefore, we hypothesize that the reduced NADPH production capacity due to the IDH1 mutation renders GBM cells more vulnerable to irradiation and chemotherapy thus prolonging survival of the patients.

Autonomous regulation of osteosarcoma cell invasiveness by Wnt5a/Ror2 signaling.

The receptor tyrosine kinase Ror2 regulates cell migration by acting as a receptor or co-receptor for Wnt5a. Although Wnt5a has been implicated in the invasiveness of several types of tumors, the role of Ror2 in tumor invasion remains elusive. Here we show that osteosarcoma cell lines SaOS-2 and U2OS show invasive properties in vitro by activating Wnt5a/Ror2 signaling in a cell-autonomous manner. The suppressed expression of either Wnt5a or Ror2 in osteosarcoma cells inhibits cell invasiveness accompanying decreased invadopodia formation. Gene-expression profiling identified matrix metalloproteinase 13 (MMP-13) as one of the genes whose expression is downregulated in SaOS-2 cells following suppression of Ror2 expression. Reduced expression or activity of MMP-13 suppresses invasiveness of SaOS-2 cells. Moreover, expression of MMP-13 and cell invasiveness by Wnt5a/Ror2 signaling can be abrogated by an inhibitor of the Src-family protein tyrosine kinases (SFKs), suggesting the role of the SFKs in MMP-13 expression through Wnt5a/Ror2 signaling. We further show that activation of an SFK is inhibited by the suppressed expression of Ror2. Collectively, these results indicate that Wnt5a/Ror2 signaling involves the activation of a SFK, leading to MMP-13 expression, and that constitutively active Wnt5a/Ror2 signaling confers invasive properties on osteosarcoma cells in a cell-autonomous manner.

Inhibition of Wnt signaling by ICAT, a novel beta-catenin-interacting protein.

Wnt signaling has an important role in both embryonic development and tumorigenesis. beta-Catenin, a key component of the Wnt signaling pathway, interacts with the TCF/LEF family of transcription factors and activates transcription of Wnt target genes. Here, we identify a novel beta-catenin-interacting protein, ICAT, that was found to inhibit the interaction of beta-catenin with TCF-4 and represses beta-catenin-TCF-4-mediated transactivation. Furthermore, ICAT inhibited Xenopus axis formation by interfering with Wnt signaling. These results suggest that ICAT negatively regulates Wnt signaling via inhibition of the interaction between beta-catenin and TCF and is integral in development and cell proliferation.

Interaction between Wnt and TGF-beta signalling pathways during formation of Spemann's organizer.

Members of the Wnt and TGF-beta superfamilies regulate both cell fate and proliferation during development and tissue maintenance. In the early amphibian embryo, the Wnt and TGF-beta superfamily signalling cascades are required for the establishment of a dorsal signalling centre, Spemann's organizer. Intracellular proteins of both pathways, upon activation, translocate to the nucleus to participate in transcription. Here we show that beta-catenin and Lef1/Tcf, which are downstream components of the Wnt signalling cascade, form a complex with Smad4, an essential mediator of signals initiated by members of the TGF-beta growth factor superfamily. In Xenopus, this interaction directly and synergistically affects expression of the twin (Xtwn) gene during formation of the organizer. This is, to our knowledge, the first demonstration of a physical interaction between TGF-beta and Wnt signalling components in vivo.

Smad8B, a Smad8 splice variant lacking the SSXS site that inhibits Smad8-mediated signalling.

BACKGROUND: Members of the TGF-beta superfamily of ligands bind to and activate surface serine/threonine-kinase receptors. Transduction of these signals requires the Smad proteins, which transiently interact with the activated receptor complex and are phosphorylated on their C terminus, SSXS site, by the type I receptor. Smad8 is a downstream signalling mediator of ALK2/ActRIA. RESULTS: We have cloned a splice variant of Smad8, designated Smad8B. The Smad8 and Smad8B cDNAs are identical in sequence, except that Smad8B lacks a portion encoding 47 amino acids, including the SSXS phosphorylation site, in the C-terminal MH2 region. Both Smad8 and Smad8B were expressed in many of the same cell types. Smad8B was capable of specific complex formation with either Smad8 or Smad4 in mammalian cells. In cells expressing constitutively activated ALK2, Smad8B was localized to the cytoplasmic region, whereas Smad8 was translocated into the nucleus. In mammalian cells, Smad8B acted as a dominant inhibitor of BMP signalling. CONCLUSIONS: Smad8B, a splice variant of Smad8, was isolated and found to specifically associate with both Smad8 and Smad4. Smad8B inhibited BMP signalling. Smad8 and Smad8B thus represent novel signal transduction proteins that may regulate the BMP signalling pathway.

Involvement of the p38 mitogen-activated protein kinase pathway in transforming growth factor-beta-induced gene expression.

Transforming growth factor-beta (TGF-beta)-activated kinase 1 (TAK1), a member of the mitogen-activated protein kinase kinase kinase family, is suggested to be involved in TGF-beta-induced gene expression, but the signaling mechanism from TAK1 to the nucleus remains largely undefined. We have found that p38 mitogen-activated protein kinase, and its direct activator MKK6 are rapidly activated in response to TGF-beta. Expression of dominant negative MKK6 or dominant negative TAK1 inhibited the TGF-beta-induced transcriptional activation as well as the p38 activation. Constitutive activation of the p38 pathway in the absence of TGF-beta induced the transcriptional activation, which was enhanced synergistically by coexpression of Smad2 and Smad4 and was inhibited by expression of the C-terminal truncated, dominant negative Smad4. Furthermore, we have found that activating transcription factor-2 (ATF-2), which is known as a nuclear target of p38, becomes phosphorylated in the N-terminal activation domain in response to TGF-beta, that ATF-2 forms a complex with Smad4, and that the complex formation is enhanced by TGF-beta. In addition, expression of a nonphosphorylatable form of ATF-2 inhibited the TGF-beta-induced transcriptional activation. These results show that the p38 pathway is activated by TGF-beta and is involved in the TGF-beta-induced transcriptional activation by regulating the Smad-mediated pathway.

The TAK1-NLK-MAPK-related pathway antagonizes signalling between beta-catenin and transcription factor TCF.

The Wnt signalling pathway regulates many developmental processes through a complex of beta-catenin and the T-cell factor/lymphoid enhancer factor (TCF/LEF) family of high-mobility-group transcription factors. Wnt stabilizes cytosolic beta-catenin, which then binds to TCF and activates gene transcription. This signalling cascade is conserved in vertebrates, Drosophila and Caenorhabditis elegans. In C. elegans, the proteins MOM-4 and LIT-1 regulate Wnt signalling to polarize responding cells during embryogenesis. MOM-4 and LIT-1 are homologous to TAK1 (a kinase activated by transforming growth factor-beta) mitogen-activated protein-kinase-kinase kinase (MAP3K) and MAP kinase (MAPK)-related NEMO-like kinase (NLK), respectively, in mammalian cells. These results raise the possibility that TAK1 and NLK are also involved in Wnt signalling in mammalian cells. Here we show that TAK1 activation stimulates NLK activity and downregulates transcriptional activation mediated by beta-catenin and TCF. Injection of NLK suppresses the induction of axis duplication by microinjected beta-catenin in Xenopus embryos. NLK phosphorylates TCF/LEF factors and inhibits the interaction of the beta-catenin-TCF complex with DNA. Thus, the TAK1-NLK-MAPK-like pathway negatively regulates the Wnt signalling pathway.

XIAP, a cellular member of the inhibitor of apoptosis protein family, links the receptors to TAB1-TAK1 in the BMP signaling pathway.

Signals elicited by transforming growth factor-beta (TGF-beta) superfamily ligands are generated following the formation of heteromeric receptor complexes consisting of type I and type II receptors. TAK1, a member of the MAP kinase kinase kinase family, and its activator, TAB1, participate in the bone morphogenetic protein (BMP) signaling pathway involved in mesoderm induction and patterning in early Xenopus embryos. However, the events leading from receptor activation to TAK1 activation remain to be identified. A yeast interaction screen was used to search for proteins that function in the pathway linking the receptors and TAB1-TAK1. The human X-chromosome-linked inhibitor of apoptosis protein (XIAP) was isolated as a TAB1-binding protein. XIAP associated not only with TAB1 but also with the BMP receptors in mammalian cells. Injection of XIAP mRNA into dorsal blastomeres enhanced the ventralization of Xenopus embryos in a TAB1-TAK1-dependent manner. Furthermore, a truncated form of XIAP lacking the TAB1-binding domain partially blocked the expression of ventral mesodermal marker genes induced by a constitutively active BMP type I receptor. These results suggest that XIAP participates in the BMP signaling pathway as a positive regulator linking the BMP receptors and TAB1-TAK1.

Nuclear translocation and increased expression of Bax and disturbance in cell cycle progression without prominent apoptosis induced by hyperthermia.

Effects of hyperthermia at 42.5 degreesC for 6 h on cell survival, cell cycle progression, and the localization and expression levels of Bcl-2 and Bax, as well as the association between Bcl-2 and Bax in human lung cancer cells were investigated. Untreated human lung cancer cells, though immortalized, expressed Bax unlike peripheral lymphocytes with low Bax expression. Bcl-2 was localized only in the cytoplasm in all the cell lines tested, whereas Bax was localized in the cytoplasm and/or nucleus; (1) only in the nucleus in three cell lines, (2) either in the nucleus or the cytoplasm in three cell lines, (3) in both the nucleus and the cytoplasm in one cell line, and (4) only in the cytoplasm in three cell lines. Of 10 cell lines examined, 6 had a low sensitivity to hyperthermia with a viability of 50% or more, and four cell lines had a high sensitivity to hyperthermia with a viability of less than 50% regardless of cell type. In cell lines highly sensitive to hyperthermia, Bax was localized in the nucleus. Hyperthermia increased the cellular level of Bax, but not Bcl-2, and reduced the association between Bcl-2 and Bax expression in PC-10 cells. Although the Bax level increased, hyperthermia induced only mild apoptosis and caused prominent cell cycle disturbance, especially in the S and G2M phases. Thus, hyperthermia at 42.5 degreesC for 6 h had cytostatic effect as well as caused mild apoptosis. Interestingly, during 3 h of hyperthermia, Bax translocated from the cytoplasm to the nucleus, whereas Bcl-2 remained in the cytoplasm. These results raise the possibility that Bax may lose its function as the inducer of apoptosis by translocating into the nucleus or have an unknown role in the nucleus.

BRAM1, a BMP receptor-associated molecule involved in BMP signalling.

BACKGROUND: TGF-beta superfamily members elicit signals through the stimulation of serine/threonine-kinase receptors. Recently, molecules associated with several TGF-beta family receptors have been cloned. One such molecule, the immunophilin FKBP12, has been reported to interact with TGF-beta family type I receptors. However, the identity of signalling specific molecules interacting with the receptor was unknown. RESULTS: To clarify the factors mediating bone morphogenetic protein (BMP) receptor signalling, a cytoplasmic molecule associated with the BMP type IA receptor (BMPR-IA) was isolated using the yeast two-hybrid system. We designated the molecule BMP receptor associated molecule 1 (BRAM1). BRAM1 is an alternatively spliced form of BS69, a factor previously identified as an adenovirus E1A-associated protein. BRAM1 was localized to the cytoplasmic region in mammalian cells, whereas BS69 is localized to the nucleus. BRAM1 bound specifically to BMPR-IA in mammalian cells. The C-terminal half of BRAM1 was found to be sufficient for binding to BMPR-IA. CONCLUSIONS: BRAM1, a BMPR-IA associated molecule, was isolated using the yeast two-hybrid system, and found to associate specifically with BMPR-IA. BRAM1 may thus serve as an interacting protein in the BMP signal pathway.