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Background: A total hip arthroplasty (THA) is a successful and reliable operation with few complications. These complications however, do form a potential source for compensation claims. In the Netherlands, there are no studies available concerning filed claims after THA. The aim of this study was to determine the incidence of claims related to THAs in the Netherlands and the reasons to claim, which claims lead to compensation, the costs involved for the insurer, and the demographics of the claimants., Methods: In this observational study, we analyzed all closed claims from 2000 to 2012 from the national largest insurer of medical liability and compared it to data from our national implant registry in the Netherlands. With the intention to contribute to prevention, we have identified the demographics of the claimant, the reasons for filing claims, and the outcome of claims., Results: Overall, 516 claims were expressed in 280 closed claim files after THA. Claims were most often related to sciatic nerve injury (19.6%). Most claimants were women (71.6%) with an average age of 63.1 years. The median cost per compensated claim is €5.921., Conclusion: The claimant is more likely to be female and to be younger than the average patient receiving a THA. The incidence of a claim after a THA is 0.14%-0.30%. Nerve damage is the most common reason to file for compensation. The distribution in reasons to claim does not resemble the complication rate in literature after a THA. The outcome of this study can be used to improve patient care, safety, and costs., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The aim was to identify mutations in serum cell-free DNA (cfDNA) associated with disease progression on tamoxifen treatment in metastatic breast cancer (MBC). Sera available at start of therapy, during therapy and at disease progression were selected from 10 estrogen receptor (ER)-positive breast cancer patients. DNA from primary tumor and normal tissue and cfDNA from minute amounts of sera were analyzed by targeted next generation sequencing (NGS) of 45 genes (1,242 exons). At disease progression, stop-gain single nucleotide variants (SNVs) for CREBBP (1 patient) and SMAD4 (1 patient) and non-synonymous SNVs for AKAP9 (1 patient), PIK3CA (2 patients) and TP53 (2 patients) were found. Mutations in CREBBP and SMAD4 have only been occasionally reported in breast cancer. All mutations, except for AKAP9, were also present in the primary tumor but not detected in all blood specimens preceding progression. More sensitive detection by deeper re-sequencing and digital PCR confirmed the occurrence of circulating tumor DNA (ctDNA) and these biomarkers in blood specimens., Competing Interests: None of the authors has a conflict of interest.

Acute renal failure, a serious condition characterised by a drastic decline in renal function, often follows ischaemia/reperfusion (I/R) episodes. I/R is characterised by necrosis, inflammation and activation of coagulation, in concert causing renal tissue damage. In this context, activated protein C (APC) might be of importance in the pathogenesis of renal I/R. APC is a serine protease which has anticoagulant but also several anti-inflammatory and cytoprotective effects such as protection of endothelial barrier function. It was our objective to study the role of cytoprotective and anticoagulant functions of APC during renal I/R. C57BL/6j mice subjected to renal I/R were treated with intraperitoneally injected exogenous human APC, or two mutant forms of APC (200 µg/kg) which specifically lack anticoagulant or signalling properties. In a different experiment mice received specific monoclonal antibodies (20 mg/kg) that block the cytoprotective and/or anticoagulant properties of endogenous APC. Treatment with APC reduced tubular injury and enhanced renal function without altering the inflammatory response and did reduce renal fibrin deposition. Administration of APC mutant lacking anticoagulant properties reduced renal damage and enhanced renal function. Blocking the anticoagulant and cytoprotective functions of endogenous APC resulted in elevated tubular damage and reduced tubular cell proliferation, however, without influencing renal function or the inflammatory response. Furthermore, blocking both the anticoagulant and cytoprotective effects of APC resulted in dramatic renal interstitial haemorrhage, indicative of impaired vascular integrity. Blocking only the anticoagulant function of APC did not result in interstitial bleeding. In conclusion, the renoprotective effect of APC during I/R is independent of its anticoagulant properties.

Background: Molecular characterization of circulating tumor cells (CTC) is promising for personalized medicine. We aimed to identify a CTC gene expression profile predicting outcome to first-line aromatase inhibitors in metastatic breast cancer (MBC) patients., Methods: CTCs were isolated from 78 MBC patients before treatment start. mRNA expression levels of 96 genes were measured by quantitative reverse transcriptase polymerase chain reaction. After applying predefined exclusion criteria based on lack of sufficient RNA quality and/or quantity, the data from 45 patients were used to construct a gene expression profile to predict poor responding patients, defined as disease progression or death <9 months, by a leave-one-out cross validation., Results: Of the 45 patients, 19 were clinically classified as poor responders. To identify them, the 75% most variable genes were used to select genes differentially expressed between good and poor responders. An 8-gene CTC predictor was significantly associated with outcome (Hazard Ratio [HR] 4.40, 95% Confidence Interval [CI]: 2.17-8.92, P < 0.001). This predictor identified poor responding patients with a sensitivity of 63% and a positive predictive value of 75%, while good responding patients were correctly predicted in 85% of the cases. In multivariate Cox regression analysis, including CTC count at baseline, the 8-gene CTC predictor was the only factor independently associated with outcome (HR 4.59 [95% CI: 2.11-9.56], P < 0.001). This 8-gene signature was not associated with outcome in a group of 71 MBC patients treated with systemic treatments other than AI., Conclusions: An 8-gene CTC predictor was identified which discriminates good and poor outcome to first-line aromatase inhibitors in MBC patients. Although results need to be validated, this study underscores the potential of molecular characterization of CTCs.

Our previous study demonstrated that high mRNA levels for Seven in Absentia Homolog 2 (SIAH2) correlated with high Estrogen Receptor (ER) mRNA levels and with longer progression-free survival (PFS) after first-line tamoxifen. Others showed high SIAH2 protein levels in ER-negative breast cancer associated with an unfavorable relapse-free survival. In the current study, we investigated SIAH2 protein expression to clarify the discrepancy between protein and mRNA findings and to determine its diagnostic value in breast cancer patients. Tissue microarrays (TMAs) containing core specimens of primary breast tumors were immunohistochemically stained for SIAH2 protein. The TMAs analyzed a cohort of 746 patients with primary breast cancer (PBC) and a cohort of 245 patients with ER-positive metastatic breast cancer (MBC) treated with first-line tamoxifen. SIAH2 staining was scored for intensity and proportion of positive tumor cells and evaluated for its relationship with metastasis-free survival (MFS) and PFS. Multivariate survival analyses included traditional prognostic or predictive factors, respectively. The PBC-cohort had 263 patients with high SIAH2 protein expression and decreased expression of ER protein and mRNA levels (P = 0.005 and P = 0.003, respectively). High SIAH2 levels correlated with significant unfavorable MFS in lymph node negative, ER-positive breast cancer patients. The MBC-cohort had 86 patients with increased SIAH2 protein expression. High SIAH2 expression was associated with an unfavorable PFS after first-line tamoxifen in multivariate analyses (HR = 1.45; 95% CI, 1.07-1.96; P = 0.015). In conclusion, SIAH2 protein expression is especially observed in ER-negative tumors. Its prognostic value in breast cancer does not add to current prognostic markers. The proportion of SIAH2-positive cells can be used as biomarker to predict tamoxifen treatment failure in MBC patients.

Background: Beside their role in hemostasis, platelets serve as sentinel cells in host defense during infection. In sepsis, platelets have been implicated in both beneficial (antibacterial) and detrimental responses (thrombosis and organ damage). Toll-like receptors and their common adaptor, myeloid differentiation factor 88 (MyD88), are essential for pathogen recognition and protective immunity. Platelets express functional Toll-like receptors and MyD88, which participate in platelet responsiveness to bacterial agonists., Objective: Considering the pivotal involvement of platelets and MyD88 in the host response to bacteria, we studied the role of platelet MyD88 in gram-negative sepsis using intravenous and airway infections with the common human sepsis pathogen Klebsiella pneumoniae., Methods: Platelet-specific Myd88(-/-) mice were generated by crossing mice with a conditional Myd88 flox allele with mice expressing Cre recombinase controlled by the platelet factor 4 promoter. In a reverse approach, full Myd88(-/-) mice were transfused with wild-type platelets., Results: In both settings, platelet MyD88 did not impact on bacterial growth or dissemination. In addition, platelet MyD88 did not influence hallmark sepsis responses such as thrombocytopenia, coagulation or endothelial activation, or distant organ injury. Platelet MyD88 played no role in lung pathology during pneumonia-derived sepsis., Conclusion: Despite known literature, platelet MyD88-dependent TLR signaling does not contribute to the host response during gram-negative sepsis., (© 2015 International Society on Thrombosis and Haemostasis.)

Background: Patients with Estrogen Receptor α-positive (ER+) Inflammatory Breast Cancer (IBC) are less responsive to endocrine therapy compared with ER+ non-IBC (nIBC) patients. The study of ER+ IBC samples might reveal biomarkers for endocrine resistant breast cancer., Materials & Methods: Gene expression profiles of ER+ samples from 201 patients were explored for genes that discriminated between IBC and nIBC. Classifier genes were applied onto clinically annotated expression data from 947 patients with ER+ breast cancer and validated with RT-qPCR for 231 patients treated with first-line tamoxifen. Relationships with metastasis-free survival (MFS) and progression-free survival (PFS) following adjuvant and first-line endocrine treatment, respectively, were investigated using Cox regression analysis., Results: A metagene of six genes including the genes encoding for 4-aminobutyrate aminotransferase (ABAT) and Stanniocalcin-2 (STC2) were identified to distinguish 22 ER+ IBC from 43 ER+ nIBC patients and remained discriminatory in an independent series of 136 patients. The metagene and two genes were not prognostic in 517 (neo)adjuvant untreated lymph node-negative ER+ nIBC breast cancer patients. Only ABAT was related to outcome in 250 patients treated with adjuvant tamoxifen. Three independent series of in total 411 patients with advanced disease showed increased metagene scores and decreased expression of ABAT and STC2 to be correlated with poor first-line endocrine therapy outcome. The biomarkers remained predictive for first-line tamoxifen treatment outcome in multivariate analysis including traditional factors or published signatures. In an exploratory analysis, ABAT and STC2 protein expression levels had no relation with PFS after first-line tamoxifen., Conclusions: This study utilized ER+ IBC to identify a metagene including ABAT and STC2 as predictive biomarkers for endocrine therapy resistance., (Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Introduction: Endocrine therapies targeting cell proliferation and survival mediated by estrogen receptor α (ERα) are among the most effective systemic treatments for ERα-positive breast cancer. However, most tumors initially responsive to these therapies acquire resistance through mechanisms that involve ERα transcriptional regulatory plasticity. Herein we identify VAV3 as a critical component in this process., Methods: A cell-based chemical compound screen was carried out to identify therapeutic strategies against resistance to endocrine therapy. Binding to ERα was evaluated by molecular docking analyses, an agonist fluoligand assay and short hairpin (sh)RNA-mediated protein depletion. Microarray analyses were performed to identify altered gene expression. Western blot analysis of signaling and proliferation markers, and shRNA-mediated protein depletion in viability and clonogenic assays, were performed to delineate the role of VAV3. Genetic variation in VAV3 was assessed for association with the response to tamoxifen. Immunohistochemical analyses of VAV3 were carried out to determine its association with therapeutic response and different tumor markers. An analysis of gene expression association with drug sensitivity was carried out to identify a potential therapeutic approach based on differential VAV3 expression., Results: The compound YC-1 was found to comparatively reduce the viability of cell models of acquired resistance. This effect was probably not due to activation of its canonical target (soluble guanylyl cyclase), but instead was likely a result of binding to ERα. VAV3 was selectively reduced upon exposure to YC-1 or ERα depletion, and, accordingly, VAV3 depletion comparatively reduced the viability of cell models of acquired resistance. In the clinical scenario, germline variation in VAV3 was associated with the response to tamoxifen in Japanese breast cancer patients (rs10494071 combined P value = 8.4 × 10-4). The allele association combined with gene expression analyses indicated that low VAV3 expression predicts better clinical outcome. Conversely, high nuclear VAV3 expression in tumor cells was associated with poorer endocrine therapy response. Based on VAV3 expression levels and the response to erlotinib in cancer cell lines, targeting EGFR signaling may be a promising therapeutic strategy., Conclusions: This study proposes VAV3 as a biomarker and a rationale for its use as a signaling target to prevent and/or overcome resistance to endocrine therapy in breast cancer.

Aromatase inhibitors are the major first-line treatment of estrogen receptor-positive breast cancer, but resistance to treatment is common. To date, no biomarkers have been validated clinically to guide subsequent therapy in these patients. In this study, we mapped the genome-wide chromatin-binding profiles of estrogen receptor α (ERα), along with the epigenetic modifications H3K4me3 and H3K27me3, that are responsible for determining gene transcription (n = 12). Differential binding patterns of ERα, H3K4me3, and H3K27me3 were enriched between patients with good or poor outcomes after aromatase inhibition. ERα and H3K27me3 patterns were validated in an additional independent set of breast cancer cases (n = 10). We coupled these patterns to array-based proximal gene expression and progression-free survival data derived from a further independent cohort of 72 aromatase inhibitor-treated patients. Through this approach, we determined that the ERα and H3K27me3 profiles predicted the treatment outcomes for first-line aromatase inhibitors. In contrast, the H3K4me3 pattern identified was not similarly informative. The classification potential of these genes was only partially preserved in a cohort of 101 patients who received first-line tamoxifen treatment, suggesting some treatment selectivity in patient classification., (©2013 AACR)

PIK3CA mutations occur frequently in breast cancer, predominantly in exons 9 and 20. The aim of this retrospective study is to evaluate the PIK3CA mutation status for its relationship with prognosis and first-line endocrine therapy outcome. PIK3CA exon 9 and 20 were evaluated for mutations in 1,352 primary breast cancer specimens by SnaPshot multiplex analyses. The mutation status was studied for their relationship with metastasis-free survival (MFS) in 342 untreated lymph node-negative (LNN) patients and to time to progression (TTP) in estrogen receptor (ER)-positive patients with metastatic disease treated with first-line tamoxifen (N = 447) or aromatase inhibitors (AIs; N = 84). We detected in 423 patients hotspot mutations for PIK3CA (31 %). Mutations in exon 20 were detected in 251 patients (59 %), with H1047L and H1047R mutations in 37 (15 %) and 214 (85 %) cases, respectively. Mutations in PIK3CA exon 9 were discovered in 173 patients (41 %), with E542K and E545K mutations in 57 (32 %) and 104 (60 %) cases as most prevalent ones. Evaluation of the untreated LNN patients for prognosis showed no relationship between MFS and PIK3CA mutations, neither for exon 9 [HR = 1.04 (95 % CI 0.57-1.89), P = 0.90] nor for exon 20 [HR = 0.98 (95 % CI 0.63-1.54); P = 0.94] when compared to wild-type. The PIK3CA mutation status was also not associated with treatment outcome after first-line tamoxifen. On the other hand, patients treated with first-line AIs showed a longer TTP when having a PIK3CA mutation in exon 9 [HR = 0.40 (95 % CI 0.17-0.95); P = 0.038] or exon 20 [HR = 0.50 (95 % CI 0.27-0.91); P = 0.024] compared to wild-types, both significant in uni- and multivariate analysis including traditional predictive factors. All results remained when only HER2-negative patients were evaluated for each cohort. PIK3CA mutations in ER-positive tumors were significantly associated with a favorable outcome after first-line AIs, which needs further confirmation in other datasets. Mutations were not associated with prognosis in untreated LNN patients nor predictive outcome after first-line tamoxifen therapy in advanced disease patients.

Background: Breast cancer patients with node positive disease can have an excellent outcome with tamoxifen only. It is unclear whether analysing both the 70-gene signature and hormone receptors provides superior prediction of outcome in tamoxifen-treated patients than either alone., Methods: Three series were evaluated: 121 patients (81% node positive) received adjuvant tamoxifen, 151 patients did not receive tamoxifen (10% node positive) and 92 patients received tamoxifen for metastatic disease. The 70-gene signature was analysed using MammaPrint. Oestrogen receptor (ER) and progesterone receptor (PR) immunohistochemistry was evaluated following St. Gallen Consensus (Highly Endocrine Responsive: ER and PR ≥ 50%, Incompletely Endocrine Responsive: ER and/or PR low or either one absent)., Results: In patients treated with adjuvant tamoxifen, both the 70-gene signature (adjusted for Endocrine Response Categories HR 2.17, 95%CI 1.01-4.66) as well as the Endocrine Response Categories (adjusted for 70-gene signature HR 6.35, 95%CI 1.90-21.3) were associated with breast-cancer-specific-survival (BCSS). Also in patients treated with tamoxifen for metastatic disease, combined analysis of the 70-gene signature and ER/PR revealed additional value (multivariate Cox regression, p = 0.013). In patients who did not receive tamoxifen, only the 70-gene signature was associated with outcome., Conclusion: In the series analysed, the 70-gene signature was mainly a prognostic factor, while ER and PR levels were mainly associated with outcome after tamoxifen. Combination of these three factors may improve outcome prediction in tamoxifen-treated patients., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

For patients with metastatic breast cancer, we previously described that increased EZH2 expression levels were associated with an adverse outcome to tamoxifen therapy. Main objective of the present study is to investigate miR-26a and miR-101 levels, which both target EZH2, for their association with molecular pathways and with efficacy of tamoxifen as first-line monotherapy for metastatic breast cancer. Expression levels were measured using quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) in primary breast cancer specimens of 235 estrogen receptor-α (ER)-positive patients. Pathway analysis was performed on microarray data available for 65 of these tumors. Logistic regression and Cox uni- and multivariate analysis were performed to relate expression levels with clinical benefit and time to progression (TTP). Increasing levels of miR-26a were significantly (P < 0.005) associated with both clinical benefit and prolonged TTP, whereas miR-101 was not. Cell cycle regulation and CCNE1 and CDC2 were the only significant overlapping pathway and genes differentially expressed between tumors with high and low levels of miR-26a and EZH2, respectively. In addition, increasing mRNA levels of CCNE1 (P < 0.05) and CDC2 (P < 0.001) were related to poor outcome. Multivariate analysis revealed miR-26a and CDC2 as an optimal set of markers associated with outcome on tamoxifen therapy, independently of traditional predictive factors. To summarize, only miR-26a levels are related with treatment outcome. Cell cycle regulation is the only overlapping pathway linked to miR-26a and EZH2 levels. Low mRNA levels of EZH2, CCNE1, and CDC2, and high levels of miR-26a are associated with favorable outcome on tamoxifen.

Objective: To assess what proportion of surgical malpractice claims might be prevented by the use of a surgical safety checklist., Background: Surgical disciplines are overrepresented in the distribution of adverse events. The recently described multidisciplinary SURgical PAtient Safety System (SURPASS) checklist covers the entire surgical pathway from admission to discharge and is being validated in various ways. Malpractice claims constitute an important source of information on adverse events. In this study, surgical malpractice claims were evaluated in detail to assess the proportion and nature of claims that might have been prevented if the SURPASS checklist had been used., Methods: A retrospective claim record review was performed using the database of the largest Dutch insurance company for medical liability. All accepted or settled closed surgical malpractice claims filed as a consequence of an incident that occurred between January 1, 2004 and December 31, 2005 were included. Data on the type and outcome of the incident and contributing factors were extracted. All contributing factors were compared to the SURPASS checklist to assess which incidents the checklist might have prevented., Results: We included 294 claims. Failure in diagnosis and peroperative damage were the most common types of incident; cognitive contributing factors were present in two-thirds of claims. Of a total of 412 contributing factors, 29% might have been intercepted by the SURPASS checklist. The checklist might have prevented 40% of deaths and 29% of incidents leading to permanent damage., Conclusion: Nearly one-third of all contributing factors in accepted surgical malpractice claims of patients that had undergone surgery might have been intercepted by using a comprehensive surgical safety checklist. A considerable amount of damage, both physical and financial, is likely to be prevented by using the SURPASS checklist.

The purpose of this study is to investigate EZH2 in a large series of breast cancer patients for its prognostic and predictive value, and to evaluate its functional role in treatment response in vitro. EZH2 levels were measured using quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) in primary breast cancer specimens and related to clinicopathologic factors and disease outcome. EZH2 expression was downregulated with siRNAs in MCF7, to assess expression alterations of putative EZH2 downstream genes and to determine cell numbers after treatment with the anti-estrogen ICI 164384. In 688 lymph node-negative patients who did not receive adjuvant systemic therapy, EZH2 was not significantly correlated with metastasis-free survival (MFS). In 278 patients with advanced disease treated with first-line tamoxifen monotherapy, the tertile with highest EZH2 levels was associated with the lowest clinical benefit (OR = 0.48; P = 0.02) and with a shorter progression-free survival (PFS) in both univariate (HR = 1.80; P < 0.001) and multivariate analysis, including traditional factors (HR = 1.61; P = 0.004). In vitro, EZH2 silencing in MCF7 caused a 38% decrease in cell numbers (P < 0.001) whereas ICI 164384 treatment resulted in a 25% decrease (P < 0.001) compared to controls. Combining EZH2 silencing with ICI treatment reduced cell numbers with 67% (P < 0.001) compared to control conditions. EZH2 downregulation was associated with an almost two-fold upregulation of the estrogen receptor alpha (ER) (P = 0.001). In conclusion, EZH2 has no prognostic value in breast cancer. High levels of EZH2 are associated with poor outcome to tamoxifen therapy in advanced breast cancer. Downregulated EZH2 leads to upregulation of the ER and better response to anti-estrogens.

Objective: Ovarian cancer is the leading cause of death from gynecological cancers in the Western world (Parkin et al., 2005). The overall 5-year survival is only 30% (Moss and Kaye, 2002), which is for a significant part due to platinum-based chemotherapy resistance. In this study, we performed a pathway analysis on nine published gene sets associated with platinum resistance in ovarian cancer, including a study by us. With this exploratory study, we aim to identify overlapping pathways associated with platinum-based chemotherapy resistance mechanisms in ovarian cancer., Methods: Gene Ontology (GO) analysis and Ingenuity Pathway Analysis (IPA) were performed to determine which functional processes were differentially represented in the combined gene lists of nine studies (457 genes) compared to all Unigene identifiers or the Ingenuity knowledge base., Results: The GO and IPA analysis resulted in the generation of 23 gene networks, and showed that 13 GO processes (>or=2 times enriched), 71 canonical pathways (p<0.05,), eight toxicity pathways (p<0.05) and 74 biological functions (p<0.005) are significantly associated with the 9-study gene set., Conclusion and Recommendations: Several pathways identified have previously been shown to be associated with therapy resistance: these include 'oxidative stress response mediated by Nrf2,' 'TP53 signaling' and 'TGFbeta signaling.' The role of TGFbeta signaling and related miRNAs identified in the network analysis in epithelial-to-mesenchymal transition (EMT) and stemness as well as the possible relation with platin-based chemotherapy resistance are further discussed in detail. We propose that future international cooperation should aim at a uniform pooled analysis of the wealth of ovarian cancer array data already available. This will enhance the power of each separate ovarian cancer study and can lead to promising results., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Epithelial ovarian cancer is the sixth most common cancer in women worldwide and the most important cause of death from gynaecological cancers in the Western world. Our explorative pathway analysis on seven published gene-sets associated with platinum resistance in ovarian cancer reveals TP53 and transforming growth factor beta as key genes. Furthermore, the extracellular matrix was associated with chemotherapy resistance in ovarian cancer as well as endocrine resistance in breast cancer. Pathway analysis again revealed transforming growth factor beta as a key gene regulating extracellular matrix gene expression. A model is presented based on literature linking transforming growth factor beta, extracellular matrix, integrin signalling, epithelial to mesenchymal transition and regulating microRNAs with a (bivalent) role in chemotherapy response.

Purpose: In our microarray analysis we observed that Seven-in-Absentia Homolog 2 (SIAH2) levels were low in estrogen receptor (ER) positive breast tumors of patients resistant to first-line tamoxifen therapy. The aim of this study was to evaluate SIAH2 for its (a) predictive/prognostic value, and (b) functional role in endocrine therapy resistance., Patients and Methods: SIAH2 expression was measured with quantitative Real-Time-PCR (qRT-PCR) in 1205 primary breast tumor specimens and related to disease outcome. The functional role of SIAH2 was determined in human breast cancer cell lines ZR-75-1, ZR/HERc, and MCF7. Cell lines were treated with estrogen (E2), anti-estrogen ICI164.384 or epidermal growth factor (EGF). Moreover, MCF7 was treated with ICI164.384 after silencing SIAH2 expression., Results: SIAH2 was not prognostic in 603 lymph node negative patients who had not received adjuvant systemic therapy. In multivariate analysis of ER-positive tumors of 235 patients with recurrent disease, SIAH2 as continuous variable, significantly predicted first-line tamoxifen treatment failure (OR = 1.48; P = 0.05) and progression-free survival (PFS) (HR = 0.79; P = 0.007). Furthermore, in primary breast cancer patients treated with adjuvant tamoxifen, SIAH2 predicted metastasis-free survival (MFS) (HR = 0.73; P = 0.005). In vitro experiments showed that SIAH2 silencing in MCF7 cells resulted in resistance to ICI164.384-treatment when compared with mock silenced cells (P = 0.008). Interestingly, in ZR cells transfected with EGFR (ZR/HERc), SIAH2 expression was induced by E2 but downregulated by EGF., Conclusion: In primary breast tumor specimens as well as in vitro low SIAH2 levels associated with resistance to endocrine therapy. Moreover, SIAH2 expression showed an opposite regulation by E2 and EGF.

Purpose Two genes, TSC22 domain family, member 1 (TSC22D1) and prosaposin (PSAP) were identified in an in vitro functional screen for genes having a causative role in tamoxifen resistance. These genes were also present in our previously established 81-gene signature for resistance to first-line tamoxifen therapy. The aim of this study was to investigate the predictive value of these genes for tamoxifen therapy failure in patients with recurrent breast cancer. Experimental Design The mRNA levels of TSC22D1 and PSAP were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) in 223 estrogen receptor-positive primary breast tumors of patients with recurrent disease treated with first-line tamoxifen therapy. The main objective of this study was the length of progression-free survival (PFS). Results High mRNA levels of TSC22D1 and PSAP were significantly associated with shorter PFS and both were independent of the traditional predictive factors (HR = 1.30, 95% CI = 1.04-1.64 P = 0.023; and HR = 1.40, 95% CI = 1.03-1.88, P = 0.029, respectively). In multivariate analysis, patients with high mRNA levels of both genes associated significantly with no clinical benefit (OR = 0.19, 95% CI = 0.06-0.62, P = 0.006) and had the shortest PFS (HR = 2.05, 95% CI = 1.29-3.25, P = 0.002). Conclusion These results confirm our previous in vitro and tumor-related findings and are indicative for the failure of tamoxifen treatment in breast-cancer patients. Both TSC22D1 and PSAP are associated with clinical outcome and may have a functional role in therapy resistance.

Background: Molecular signatures that predict outcome in tamoxifen treated breast cancer patients have been identified. For the first time, we compared these response profiles in an independent cohort of (neo)adjuvant systemic treatment naïve breast cancer patients treated with first-line tamoxifen for metastatic disease., Methods: From a consecutive series of 246 estrogen receptor (ER) positive primary tumors, gene expression profiling was performed on available frozen tumors using 44K oligoarrays (n = 69). A 78-gene tamoxifen response profile (formerly consisting of 81 cDNA-clones), a 21-gene set (microarray-based Recurrence Score), as well as the HOXB13-IL17BR ratio (Two-Gene-Index, RT-PCR) were analyzed. Performance of signatures in relation to time to progression (TTP) was compared with standard immunohistochemical (IHC) markers: ER, progesterone receptor (PgR) and HER2., Results: In univariate analyses, the 78-gene tamoxifen response profile, 21-gene set and HOXB13-IL17BR ratio were all significantly associated with TTP with hazard ratios of 2.2 (95% CI 1.3-3.7, P = 0.005), 2.3 (95% CI 1.3-4.0, P = 0.003) and 4.2 (95% CI 1.4-12.3, P = 0.009), respectively. The concordance among the three classifiers was relatively low, they classified only 45-61% of patients in the same category. In multivariate analyses, the association remained significant for the 78-gene profile and the 21-gene set after adjusting for ER and PgR., Conclusion: The 78-gene tamoxifen response profile, the 21-gene set and the HOXB13-IL17BR ratio were all significantly associated with TTP in an independent patient series treated with tamoxifen. The addition of multigene assays to ER (IHC) improves the prediction of outcome in tamoxifen treated patients and deserves incorporation in future clinical studies.

Purpose: We previously discovered an extracellular matrix (ECM) gene cluster associated with resistance to first-line tamoxifen therapy of patients with metastatic breast cancer. In this study, we determined whether the six individual ECM genes [collagen 1A1 (COL1A1), fibronectin 1 (FN1), lysyl oxidase (LOX), secreted protein acidic cysteine-rich (SPARC), tissue inhibitor of metalloproteinase 3 (TIMP3), and tenascin C (TNC)] were associated with treatment response, prognosis, or both., Experimental Design: In 1,286 primary breast tumors, mRNA expression (quantitative real-time PCR) was related to clinicopathologic factors and disease outcome in univariate and multivariate analysis including traditional factors., Results: TIMP3, FN1, LOX, and SPARC expression levels (continuous variables) were significantly associated with distant metastasis-free survival (MFS) in 680 lymph node-negative untreated patients (P<0.03). Using a calculated linear prognostic score, these patients were evenly divided into five prognostic groups with a significant difference in 10-year MFS of approximately 40% between the two extreme prognostic groups. Furthermore, high TNC expression as continuous variable was associated with (a) shorter MFS in 139 estrogen receptor-positive and lymph node-positive patients who received adjuvant tamoxifen therapy (hazard ratio, 1.53; P=0.001), and (b) no clinical benefit (odds ratio, 0.81; P=0.035) and shorter progression-free survival (hazard ratio, 1.19; P=0.002) in 240 patients in whom recurrence was treated with tamoxifen as first-line monotherapy. These results were also significant in multivariate analyses., Conclusion: FN1, LOX, SPARC, and TIMP3 expression levels are associated with the prognosis of patients with breast cancers, whereas TNC is associated with resistance to tamoxifen therapy. Further validation and functional studies are necessary to determine the use of these ECM genes in decisions regarding treatment and whether they can serve as targets for therapy.

We set out to discover ovarian cancer biomarkers useful for monitoring progression during and after chemotherapy and possibly for diagnosis. Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry was used to create serum protein profiles of ovarian cancer patients before chemotherapy or at progression (n = 51) (trial initiated by the Gynecological Cancer Cooperative Group of the European Organization for Research and Treatment of Cancer trial) that were compared with those of healthy individuals (n = 31). In addition, sera profiles from ovarian cancer patients after chemotherapy (n = 12) were compared with those of ovarian cancer patients at progression (n = 24). One of the discovered biomarkers was identified and subsequently confirmed and validated using enzyme-linked immunosorbent assay (ELISA). Eight primary (sens = 94%, spec = 97%, P < 0.0001) and seven progression tumor biomarkers (sens = 91%, spec = 97%, P < 0.0001) were discovered. In addition, we discovered eight potential progression monitoring biomarkers (sens = 75%, spec = 83%, P = 0.0008) of which one, a biomarker of 11.7 kd, was further identified as serum amyloid A1. Independent validation (ELISA) showed an elevated expression of this protein at relapse in four of the seven ovarian cancer patients tested. Combining the eight newly discovered progression monitoring biomarkers with CA125 resulted in a clear increase of the sensitivity (91-100%). These biomarkers, in combination with for instance CA125, should be validated in large ovarian cancer and control groups. The resulting multimarker assay could be suitable for disease monitoring during and after therapy and might also be useful for ovarian cancer screening.

Background: Estrogen receptor positive (ER+) breast cancers (BC) are heterogeneous with regard to their clinical behavior and response to therapies. The ER is currently the best predictor of response to the anti-estrogen agent tamoxifen, yet up to 30-40% of ER+BC will relapse despite tamoxifen treatment. New prognostic biomarkers and further biological understanding of tamoxifen resistance are required. We used gene expression profiling to develop an outcome-based predictor using a training set of 255 ER+ BC samples from women treated with adjuvant tamoxifen monotherapy. We used clusters of highly correlated genes to develop our predictor to facilitate both signature stability and biological interpretation. Independent validation was performed using 362 tamoxifen-treated ER+ BC samples obtained from multiple institutions and treated with tamoxifen only in the adjuvant and metastatic settings., Results: We developed a gene classifier consisting of 181 genes belonging to 13 biological clusters. In the independent set of adjuvantly-treated samples, it was able to define two distinct prognostic groups (HR 2.01 95%CI: 1.29-3.13; p = 0.002). Six of the 13 gene clusters represented pathways involved in cell cycle and proliferation. In 112 metastatic breast cancer patients treated with tamoxifen, one of the classifier components suggesting a cellular inflammatory mechanism was significantly predictive of response., Conclusion: We have developed a gene classifier that can predict clinical outcome in tamoxifen-treated ER+ BC patients. Whilst our study emphasizes the important role of proliferation genes in prognosis, our approach proposes other genes and pathways that may elucidate further mechanisms that influence clinical outcome and prediction of response to tamoxifen.

Purpose: A HOXB13-to-IL17BR expression ratio was previously identified to predict clinical outcome of breast cancer patients treated with adjuvant tamoxifen. However, this ratio may predict a tumor's response to tamoxifen, its intrinsic aggressiveness, or both., Patients and Methods: We have measured the HOXB13 and IL17BR expression levels by real-time polymerase chain reaction in 1,252 primary breast tumor specimens. Expression levels were normalized to housekeeper gene levels and related to clinicopathologic factors for all patients. The primary objective of this study was to determine the relationship of a HOXB13-to-IL17BR ratio with tumor aggressiveness and/or with response to tamoxifen therapy in estrogen receptor (ER) -positive disease. We selected ER-positive tumors, and clinical end points for the HOXB13-to-IL17BR ratio were disease-free survival (DFS) in patients with primary breast cancer (N = 619) and progression-free survival (PFS) in patients with recurrent breast cancer treated with first-line tamoxifen monotherapy (N = 193). The odds ratio (OR) and hazard ratio (HR) and their 95% CI were calculated, and all P values were two-sided., Results: The HOXB13-to-IL17BR ratio was significantly associated with DFS and PFS. In multivariate analysis, HOXB13-to-IL17BR ratio expression levels were associated with a shorter DFS for node-negative patients only. Corrected for traditional predictive factors, the dichotomized HOXB13-to-IL17BR ratio was the strongest predictor in multivariate analysis for a poor response to tamoxifen therapy (OR = 0.16; 95% CI, 0.06 to 0.45; P < .001) and a shorter PFS (HR = 2.97; 95% CI, 1.82 to 4.86; P < .001)., Conclusion: High HOXB13-to-IL17BR ratio expression levels associate with both tumor aggressiveness and tamoxifen therapy failure.

The aim of this study is to discover a gene set that can predict resistance to platinum-based chemotherapy in ovarian cancer. The study was performed on 96 primary ovarian adenocarcinoma specimens from 2 hospitals all treated with platinum-based chemotherapy. In our search for genes, 24 specimens of the discovery set (5 nonresponders and 19 responders) were profiled in duplicate with 18K cDNA microarrays. Confirmation was done using quantitative RT-PCR on 72 independent specimens (9 nonresponders and 63 responders). Sixty-nine genes were differentially expressed between the nonresponders (n=5) and the responders (n=19) in the discovery phase. An algorithm was constructed to identify predictive genes in this discovery set. This resulted in 9 genes (FN1, TOP2A, LBR, ASS, COL3A1, STK6, SGPP1, ITGAE, PCNA), which were confirmed with qRT-PCR. This gene set predicted platinum resistance in an independent validation set of 72 tumours with a sensitivity of 89% (95% CI: 0.68-1.09) and a specificity of 59% (95% CI: 0.47-0.71)(OR=0.09, p=0.026). Multivariable analysis including patient and tumour characteristics demonstrated that this set of 9 genes is independent for the prediction of resistance (p<0.01). The findings of this study are the discovery of a gene signature that classifies the tumours, according to their response, and a 9-gene set that determines resistance in an independent validation set that outperforms patient and tumour characteristics. A larger independent multicentre study should further confirm whether this 9-gene set can identify the patients who will not respond to platinum-based chemotherapy and could benefit from other therapies., (Copyright (c) 2005 Wiley-Liss, Inc.)

Purpose: To discover a set of markers predictive for the type of response to endocrine therapy with the antiestrogen tamoxifen using gene expression profiling., Patients and Methods: The study was performed on 112 estrogen receptor-positive primary breast carcinomas from patients with advanced disease and clearly defined types of response (ie, 52 patients with objective response v 60 patients with progressive disease) from start of first-line treatment with tamoxifen. Main clinical end points are the effects of therapy on tumor size and time until tumor progression (progression-free survival [PFS]). RNA isolated from tumor samples was amplified and hybridized to 18,000 human cDNA microarrays., Results: Using a training set of 46 breast tumors, 81 genes were found to be differentially expressed (P < or = .05) between tamoxifen-responsive and -resistant tumors. These genes were involved in estrogen action, apoptosis, extracellular matrix formation, and immune response. From the 81 genes, a predictive signature of 44 genes was extracted and validated on an independent set of 66 tumors. This 44-gene signature is significantly superior (odds ratio, 3.16; 95% CI, 1.10 to 9.11; P = .03) to traditional predictive factors in univariate analysis and also significantly related with a longer PFS in univariate (hazard ratio, 0.54; 95% CI, 0.31 to 0.94; P = .03) as well as in multivariate analyses (P = .03)., Conclusion: Our data show that gene expression profiling can be used to discriminate between breast cancer patients with progressive disease and objective response to tamoxifen. Additional studies are needed to confirm if the predictive signature might allow identification of individual patients who could benefit from other (adjuvant) endocrine therapies.

CATs are interventions that should be considered by critical care nurses when planning interventions for meeting the needs of families of critically ill patients. More research is needed on the effect of family members providing CAT to critically ill patients and what forms of CAT are most effective. Comparison studies of CAT being provided by certified providers versus family members are needed. Using CAT for family members may be useful in reducing their levels of stress and anxiety and therefore reducing negative physiologic and psychologic responses to stress.

This paper describes the first phase of the development of a Protocol based Decision Support System (PDSS) that will be linked to an Electronic Patient Record system (EPR system). The protocol system will be pro-active: the physician will be automatically prompted from the EPR of a particular patient if the protocol that applies for that patient defines it necessary. The PropeR project studies the impact of a PDSS that is linked to an EPR on daily care processes. There are two areas of research: hospital and home care. This paper describes the application in the hospital. The protocol that is being computerized is a treatment protocol for Acute Myelogenous Leukaemia (AML) that also studies treatment alternatives (conventional versus experimental treatment). This paper based AML protocol has been translated into a formal representation. The KA-tool Gaston is used to make this representation. Twenty-eight subprotocols have been organized in a hierarchical structure with three levels. One of the aims of the project is to make a representation of the AML protocol that can be used in other organizations as well. The main problem we encountered is that the representation not only contains the content of the protocol, but also aspects of application of that protocol in daily care of the hospital and aspects of support. The solution to this problem is the creation of two layers of representation: the first layer is an exact copy of the protocol and thus sharable and the second layer focuses on the support of the protocol in the daily working processes and is mainly domain specific: for the University Hospital Maastricht. At the moment, this division into two layers is being discussed.

A recent study observed that numerical chromosome abnormalities in Hodgkin's disease (HD) are detected not only in morphologically abnormal Hodgkin/Reed-Sternberg cells, but also in a fraction of morphologically normal cells. However, the phenotypic constitution of these genetically abnormal, morphologically normal cells and their relationship to the malignant Hodgkin/Reed-Sternberg cells could not be established in the earlier cases studied, because of the low frequency of these cells. The present study investigated two cases of classical Hodgkin's disease containing a relatively large population of such apparently normal cells with aberrant chromosome copy numbers. The phenotype and their position within the developmental route of the malignant compartment were examined by a combined in situ hybridization and immunocytochemistry approach. Numerical abnormalities for chromosome 1 in one case and for chromosomes X, Y, and 1 in the other case were observed not only in CD30-positive Hodgkin/Reed-Sternberg cells, but also in CD30-negative, morphologically normal cells. It was shown that these genetically aberrant cells expressed the B-cell antigen CD19, thus confirming their B-cell nature. These studies indicate a relationship between the genome aberrations in these genetically abnormal, morphologically normal B-cells and the Hodgkin/Reed-Sternberg cells, suggesting that they are progenitor cells of the malignant cell fraction., (Copyright 1999 John Wiley & Sons, Ltd.)

Hodgkin and Reed-Sternberg cells are considered to represent the malignant fraction in Hodgkin's disease. Several studies have shown that the Hodgkin and Reed-Sternberg cells are chromosomally abnormal, but genetic data about the morphologically normal cell population in Hodgkin's disease are very limited. This latter cell population has therefore been examined for chromosomal aberrations, using the in situ hybridization (ISH) procedure, making use of DNA probes for chromosomes 1, 7, 8, 9, 11, 12, 15, 17, and 18. Nuclei were isolated from freshly frozen (10 cases) and paraffin-embedded (16 cases) biopsy samples and 1000 nuclei per case were evaluated. The cases of Hodgkin's disease were compared with reactive lymph nodes, which show aberrant chromosome copy numbers in less than 1 per cent of the cells. Using strict scoring criteria, nuclei in the tumour were found to show an abnormal genotype, in the range of 1-12 per cent, with trisomies occurring most frequently. No characteristic numerical chromosome abnormality was observed. ISH on 4 microns thick paraffin sections of six cases of Hodgkin's disease revealed numerical aberrations for chromosome 1 in cells which appeared to be morphologically normal. The genomically abnormal nuclei did not differ in morphology or size from the nuclei of morphologically normal cells, but differed considerably in size when compared with the nuclei of Hodgkin/Reed-Sternberg cells after the ISH procedure. Three of these six cases revealed a population of apparently normal cells with an aberrant copy number which differed notably from the fraction observed in reactive lymph nodes. It is concluded, therefore, that a subset of morphologically normal cells, next to the Hodgkin/Reed-Sternberg cells, are chromosomally aberrant and may participate in the malignant cell fraction of Hodgkin's disease.

Aims: In order to clarify the differentiation and proliferation status of the Reed-Sternberg and Hodgkin cells we studied A and B-type lamin expression with specific monoclonal antibodies in nodular sclerosing Hodgkin's disease. Its normal counterpart, the reactive lymph node, was also examined for lamin subtype expression., Methods and Results: The CD20 positive centrocytes and centroblasts of the follicle centre in the reactive lymph nodes expressed lamin B1, but were not or only very weakly positive for lamin B2 or A-type lamin antibodies. Mantle zone lymphocytes displayed lamins B1 and B2, but were negative for A-type lamins. Furthermore, CD3- and CD20-positive lymphocytes in the medulla and paracortex lacked A-type lamins, but were positive for both B-type lamins. Finally, the proliferation marker Ki67 was mainly detected in the centroblasts, but also in a fraction of the A-type lamin negative cells in the paracortex and medulla. In Hodgkin's disease, all cells expressed lamins B1 and B2, whereas A-type lamins were primarily observed in CD30-positive Reed-Sternberg and Hodgkin cells. About 20% of the Reed-Sternberg and Hodgkin cells expressed Ki67, with co-expression of lamin A in most of these cells., Conclusions: Ki67 and A-type lamin staining were in general mutually exclusive in lymph nodes, indicating that A-type lamin positive cells are not proliferative. This suggests also that the A-type lamin expression in Reed-Sternberg and Hodgkin cells is correlated with a relatively mature phenotype of these malignant cells. However, some of these differentiated malignant cells still have a capacity to proliferate as indicated by Ki67 positivity. Our observation that lamin B2 expression in the follicle centre cells of the reactive lymph node is low or absent indicates that this lamin subtype is not always expressed in nucleated cells, which is in clear contrast to the results obtained in previous studies in other diseases and in normal tissues. Absence of lamin B2 expression may be associated with the follicle centre stage of B-cells.

We describe a fast light microscopic procedure for the simultaneous enzyme cytochemical detection of three different DNA target sequences in contrasting colors in both interphase and metaphase cell preparations. Chromosome-specific DNA probes labeled with either biotin, digoxygenin, or fluorescein were hybridized as a mixture and detected clearly and accurately by precipitates of the horseradish peroxidase-diaminobenzidine (PO-DAB, brown color), alkaline phosphatase-Fast Red (APase-Fast Red, red color), or horseradish peroxidase-tetramethylbenzidine (PO-TMB, green color) reaction, respectively. The PO-TMB reaction product was stabilized effectively by the addition of sodium tungstate to the reaction mixture, thus making the PO-TMB reaction now generally applicable to in situ hybridization (ISH). To avoid mixing of the precipitates of the two PO reactions used in the triple-color ISH method, the first detected PO activity was always completely inactivated by a mild acid treatment before the second one was applied. Finally, the cell preparations were embedded in a thin protein layer cross-linked by formaldehyde to ensure permanent stabilization of the enzyme reaction products and optimal visualization of color contrast. The triple-color ISH detection procedure could be combined with beta-galactosidase-5-bromo-4-chloro-3-indolyl-beta- D-galactoside (beta-Gal-BCIG) immunocytochemistry (ICC), leading to the simultaneous localization of multiple DNA targets and a protein target in the same cell. The described procedure may therefore be a valuable tool in the areas of cytogenetics, cell biology, and molecular pathology.