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1.  Exon-level expression analyses identify MYCN and NTRK1 as major determinants of alternative exon usage and robustly predict primary neuroblastoma outcome 
British Journal of Cancer  2012;107(8):1409-1417.
Using mRNA expression-derived signatures as predictors of individual patient outcome has been a goal ever since the introduction of microarrays. Here, we addressed whether analyses of tumour mRNA at the exon level can improve on the predictive power and classification accuracy of gene-based expression profiles using neuroblastoma as a model.
In a patient cohort comprising 113 primary neuroblastoma specimens expression profiling using exon-level analyses was performed to define predictive signatures using various machine-learning techniques. Alternative transcript use was calculated from relative exon expression. Validation of alternative transcripts was achieved using qPCR- and cell-based approaches.
Both predictors derived from the gene or the exon levels resulted in prediction accuracies >80% for both event-free and overall survival and proved as independent prognostic markers in multivariate analyses. Alternative transcript use was most prominently linked to the amplification status of the MYCN oncogene, expression of the TrkA/NTRK1 neurotrophin receptor and survival.
As exon level-based prediction yields comparable, but not significantly better, prediction accuracy than gene expression-based predictors, gene-based assays seem to be sufficiently precise for predicting outcome of neuroblastoma patients. However, exon-level analyses provide added knowledge by identifying alternative transcript use, which should deepen the understanding of neuroblastoma biology.
PMCID: PMC3494449  PMID: 23047593
exon arrays; prediction; PAM; neuroblastoma; alternative transcript use
2.  Preoperative staging of perforated diverticulitis by computed tomography scanning 
Techniques in Coloproctology  2012;16(5):363-368.
Treatment of perforated diverticulitis depends on disease severity classified according to Hinchey’s preoperative classification. This study assessed the accuracy of preoperative staging of perforated diverticulitis by computerized tomography (CT) scanning.
All patients who presented with perforated diverticulitis between 1999 and 2009 in two teaching hospitals of Rotterdam, the Netherlands, and in addition had a preoperative CT scan within 24 h before emergency surgery were included. Two radiologists reviewed all CT scans and were asked to classify the severity of the disease according to the Hinchey classification. The CT classification was compared to Hinchey’s classification at surgery.
Seventy-five patients were included, 48 of whom (64 %) were classified Hinchey 3 or 4 perforated diverticulitis during surgery. The positive predictive value of preoperative CT scanning for different stages of perforated diverticulitis ranged from 45 to 89 %, and accuracy was between 71 and 92 %. The combination of a large amount of free intra-abdominal air and fluid was strongly associated with Hinchey 3 or 4 and therefore represented a reliable indicator for required surgical treatment.
The accuracy of predicting Hinchey’s classification by preoperative CT scanning is not very high. Nonetheless, free intra-abdominal air in combination with diffuse fluid is a reliable indication for surgery as it is strongly associated with perforated diverticulitis with generalized peritonitis. In 42 % of cases, Hinchey 3 perforated diverticulitis is falsely classified as Hinchey 1 or 2 by CT scanning.
PMCID: PMC3444700  PMID: 22752330
Perforated diverticulitis; Computed tomography scanning; Hinchey classification
3.  The influence of mechanical bowel preparation in elective colorectal surgery for diverticulitis 
Techniques in Coloproctology  2012;16(4):309-314.
Mechanical bowel preparation (MBP) has been shown to have no influence on the incidence of anastomotic leakage in overall colorectal surgery. The role of MBP in elective surgery in combination with an inflammatory component such as diverticulitis is yet unclear. This study evaluates the effects of MBP on anastomotic leakage and other septic complications in 190 patients who underwent elective surgery for colonic diverticulitis.
A subgroup analysis was performed in a prior multicenter (13 hospitals) randomized trial comparing clinical outcome of MBP versus no MBP in elective colorectal surgery. Primary endpoint was the occurrence of anastomotic leakage in patients operated on for diverticulitis, and secondary endpoints were septic complications and mortality.
Out of a total of 1,354 patients, 190 underwent elective colorectal surgery (resection with primary anastomosis) for (recurrent or stenotic) diverticulitis. One hundred and three patients underwent MBP prior to surgery and 87 did not. Anastomotic leakage occurred in 7.8 % of patients treated with MBP and in 5.7 % of patients not treated with MBP (p = 0.79). There were no significant differences between the groups in septic complications and mortality.
Mechanical bowel preparation has no influence on the incidence of anastomotic leakage, or other septic complications, and may be safely omitted in case of elective colorectal surgery for diverticulitis.
PMCID: PMC3398249  PMID: 22706733
Colonic diverticulitis; Mechanical bowel preparation; Anastomotic leak; Surgical site infection
4.  Lung Transplantation for Cystic Fibrosis 
Lung transplantation is a complex, high-risk, potentially life-saving therapy for the end-stage lung disease of cystic fibrosis (CF). The decision to pursue transplantation involves comparing the likelihood of survival with and without transplantation as well as assessing the effect of wait-listing and transplantation on the patient's quality of life. Although recent population-based analyses of the US lung allocation system for the CF population have raised controversies about the survival benefits of transplantation, studies from the United Kingdom and Canada have suggested a definite survival advantage for those receiving transplants. In response to these and other controversies, leaders in transplantation and CF met together in Lansdowne, Virginia, to consider the state of the art in lung transplantation for CF in an international context, focusing on advances in surgical technique, measurement of outcomes, use of prognostic criteria, variations in local control over listing, and prioritization among the United States, Canada, the United Kingdom, and The Netherlands, patient adherence before and after transplantation and other issues in the broader context of lung transplantation. Finally, the conference members carefully considered how efforts to improve outcomes for lung transplantation for CF lung disease might best be studied. This Roundtable seeks to communicate the substance of our discussions.
PMCID: PMC2797068  PMID: 20008865
lung transplantation; survival; quality of life; study design; adherence
5.  Incidence and prognostic value of tumour cells detected by RT–PCR in peripheral blood stem cell collections from patients with Ewing tumour 
British Journal of Cancer  2006;95(10):1326-1333.
To retrospectively evaluate the incidence of tumour cell contamination of peripheral blood stem cell (PBSC) collections and to correlate these data with the clinical outcome after high-dose chemotherapy (HDCT) with stem cell rescue in patients with a high-risk Ewing tumour. Peripheral blood stem cell collections obtained from 171 patients were analysed. Tumour contamination was assessed by reverse transcriptase–polymerase chain reaction (RT–PCR). The files of 88 patients who underwent HDCT followed by PBSC reinfusion were reviewed in detail, and their outcome compared to the PBSC RT–PCR results. Seven of 88 PBSC collections (8%) contained tumour cells as detected by RT–PCR. Peripheral blood stem cells were collected after a median of five cycles of chemotherapy. No clinical factor predictive of tumour cell contamination of PBSC harvest could be identified. Event-free survival (EFS) and overall survival (OS) of the whole study population were 45.3 % and 51.8 % at 3 years from the date of the graft, respectively. Forty-five patients relapsed with a median time of 15 months after graft, only four of whom had tumour cell contamination of the PBSC harvest. Tumour cell contamination of PBSC collection is rare and does not seem to be associated with a significantly poorer EFS or OS in this high-risk population.
PMCID: PMC2360590  PMID: 17088915
ewing tumour; PBSC; tumour cell contamination; RT–PCR; outcome
6.  Preparation of a differentially expressed, full-length cDNA expression library by RecA-mediated triple-strand formation with subtractively enriched cDNA fragments. 
Nucleic Acids Research  1996;24(17):3478-3480.
We have developed a fast and general method to obtain an enriched, full-length cDNA expression library with subtractively enriched cDNA fragments. The procedure relies on RecA-mediated triple-helix formation of single-stranded cDNA fragments with a double-stranded cDNA plasmid library. The complexes were then captured from the solutions using the digoxigenin-antidigoxigenin paramagnetic beads followed by recovery of the enriched double-stranded cDNA expression library. We have observed a linear relation between the capture of full-length cDNAs in the library and the fold enrichment in the subtracted cDNA population.
PMCID: PMC146116  PMID: 8811109
7.  Activation of the E-cadherin/catenin complex in human MCF-7 breast cancer cells by all-trans-retinoic acid. 
British Journal of Cancer  1995;72(6):1447-1453.
All-trans-retinoic acid (RA), like insulin-like growth factor I (IGF-I) and tamoxifen, inhibit invasion of human MCF-7/6 mammary cancer cells in vitro. For tamoxifen and for IGF-I, activation of the invasion-suppressor function of the E-cadherin/catenin complex was shown to be the most probable mechanism of the anti-invasive action. We did a series of experiments to determine whether the anti-invasive effect of RA also implicated the invasion-suppressor E-cadherin/catenin complex. Human MCF-7/6 mammary and HCT-8/R1 colon cancer cells, both with a dysfunctional E-cadherin/catenin complex, were treated with RA and the function of the complex was evaluated through Ca(2+)-dependent fast aggregation. Fast aggregation of both MCF-7/6 and HCT-8/R1 cells was induced by 1 microM RA. This effect was abolished by antibodies against E-cadherin. RA-induced fast aggregation was not sensitive to cycloheximide, tyrosine kinase inhibitors or antibodies against IGF-I or against the IGF-I receptor. RA did not stimulate IGF-I receptor phosphorylation or alter the E-cadherin/catenin complex, as evidenced by immunoprecipitation. RA up-regulates the function of the invasion-suppressor complex E-cadherin/catenin. Its action mechanism is different from that of IGF-I. RA may act as an anti-invasive agent with unique mechanisms of action.
PMCID: PMC2034086  PMID: 8519658
8.  Insulin-like growth factor I activates the invasion suppressor function of E-cadherin in MCF-7 human mammary carcinoma cells in vitro. 
British Journal of Cancer  1993;68(2):282-289.
The calcium-dependent cell-cell adhesion molecule E-cadherin has been shown to counteract invasion of epithelial neoplastic cells. Using three monoclonal antibodies, we have demonstrated the presence of E-cadherin at the surface of human MCF-7/6 mammary carcinoma cells by indirect immunofluorescence coupled to flow cytometry and by immunocytochemistry. Nevertheless, MCF-7/6 cells failed to aggregate in a medium containing 1.25 mM CaCl2, and they were invasive after confrontation with embryonic chick heart fragments in organ culture. Treatment of MCF-7/6 cells with 0.5 microgram ml-1 insulin-like growth factor I (IGF-I) led to homotypic aggregation within 5 to 10 min and inhibited invasion in vitro during at least 8 days. The effect of IGF-I on cellular aggregation was insensitive to cycloheximide. However, monoclonal antibodies that interfered with the function of either the IGF-I receptor (alpha IR3) or E-cadherin (HECD-1, MB2) blocked the effect of IGF-I on aggregation. The effects of IGF-I on aggregation and on invasion could be mimicked by 1 microgram ml-1 insulin, but not by 0.5 microgram ml-1 IGF-II. The insulin effects were presumably not mediated by the IGF-I receptor, since they could not be blocked by an antibody against this receptor (alpha IR3). Our results indicate that IGF-I activates the invasion suppressor role of E-cadherin in MCF-7/6 cells.
PMCID: PMC1968539  PMID: 8347483
9.  FER kinase promotes breast cancer metastasis by regulating α6- and β1-integrin-dependent cell adhesion and anoikis resistance 
Oncogene  2013;32(50):5582-5592.
Metastatic breast cancer cannot be treated successfully. Currently, the targeted therapies for metastatic disease are limited to human epidermal growth factor receptor 2 and hormone receptor antagonists. Understanding the mechanisms of breast cancer growth and metastasis is therefore crucial for the development of new intervention strategies. Here, we show that FER kinase (FER) controls migration and metastasis of invasive human breast cancer cell lines by regulating α6- and β1-integrin-dependent adhesion. Conversely, the overexpression of FER in non-metastatic breast cancer cells induces pro-invasive features. FER drives anoikis resistance, regulates tumour growth and is necessary for metastasis in a mouse model of human breast cancer. In human invasive breast cancer, high FER expression is an independent prognostic factor that correlates with high-grade basal/triple-negative tumours and worse overall survival, especially in lymph node-negative patients. These findings establish FER as a promising target for the prevention and inhibition of metastatic breast cancer.
PMCID: PMC3898493  PMID: 23873028
breast cancer; cell adhesion; cell migration; FER kinase; metastasis

Results 1-9 (9)