Most sporadic colorectal cancers are initiated by activating Wnt pathway mutations, characterized by the stabilization of beta-catenin and constitutive transcription by the beta-catenin/T cell factor-4 (Tcf-4) complex. EphB guidance receptors are Tcf4 target genes that control intestinal epithelial architecture through repulsive interactions with Ephrin-B ligands. Here we show that, although Wnt signalling remains constitutively active, most human colorectal cancers lose expression of EphB at the adenoma-carcinoma transition. Loss of EphB expression strongly correlates with degree of malignancy. Furthermore, reduction of EphB activity accelerates tumorigenesis in the colon and rectum of Apc(Min/+) mice, and results in the formation of aggressive adenocarcinomas. Our data demonstrate that loss of EphB expression represents a critical step in colorectal cancer progression.

The Netherlands launched a nationwide implementation study on non-invasive prenatal testing (NIPT) as a first-tier test offered to all pregnant women. This started on April 1, 2017 as the TRIDENT-2 study, licensed by the Dutch Ministry of Health. In the first year, NIPT was performed in 73,239 pregnancies (42% of all pregnancies), 7,239 (4%) chose first-trimester combined testing, and 54% did not participate. The number of trisomies 21 (239, 0.33%), 18 (49, 0.07%), and 13 (55, 0.08%) found in this study is comparable to earlier studies, but the Positive Predictive Values (PPV)-96% for trisomy 21, 98% for trisomy 18, and 53% for trisomy 13-were higher than expected. Findings other than trisomy 21, 18, or 13 were reported on request of the pregnant women; 78% of women chose to have these reported. The number of additional findings was 207 (0.36%); these included other trisomies (101, 0.18%, PPV 6%, many of the remaining 94% of cases are likely confined placental mosaics and possibly clinically significant), structural chromosomal aberrations (95, 0.16%, PPV 32%,) and complex abnormal profiles indicative of maternal malignancies (11, 0.02%, PPV 64%). The implementation of genome-wide NIPT is under debate because the benefits of detecting other fetal chromosomal aberrations must be balanced against the risks of discordant positives, parental anxiety, and a potential increase in (invasive) diagnostic procedures. Our first-year data, including clinical data and laboratory follow-up data, will fuel this debate. Furthermore, we describe how NIPT can successfully be embedded into a national screening program with a single chain for prenatal care including counseling, testing, and follow-up.

ObjectiveTo evaluate the clinical impact of nationwide implementation of genome‐wide non‐invasive prenatal testing (NIPT) in pregnancies at increased risk for fetal trisomies 21, 18 and 13 (TRIDENT study).MethodWomen with elevated risk based on first trimester combined testing (FCT ≥ 1:200) or medical history, not advanced maternal age alone, were offered NIPT as contingent screening test, performed by Dutch University Medical laboratories. We analyzed uptake, test performance, redraw/failure rate, turn‐around time and pregnancy outcome.ResultsBetween 1 April and 1 September 2014, 1413/23 232 (6%) women received a high‐risk FCT result. Of these, 1211 (85.7%) chose NIPT. One hundred seventy‐nine women had NIPT based on medical history. In total, 1386/1390 (99.7%) women received a result, 6 (0.4%) after redraw. Mean turn‐around time was 14 days. Follow‐up was available in 1376 (99.0%) pregnancies. NIPT correctly predicted 37/38 (97.4%) trisomies 21, 18 or 13 (29/30, 4/4 and 4/4 respectively); 5/1376 (0.4%) cases proved to be false positives: trisomies 21 (n = 2), 18 (n = 1) and 13 (n = 2). Estimated reduction in invasive testing was 62%.ConclusionIntroduction of NIPT in the Dutch National healthcare‐funded Prenatal Screening Program resulted in high uptake and a vast reduction of invasive testing. Our study supports offering NIPT to pregnant women at increased risk for fetal trisomy. © 2016 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd. © 2016 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd.

Nonsteroidal anti-inflammatory drugs (NSAIDs) have chemopreventive potential against colorectal carcinomas (CRCs). Inhibition of cyclooxygenase (COX)-2 underlies part of this effect, although COX-2-independent mechanisms may also exist. Nonsteroidal antiinflammatory drugs appear to inhibit the initial stages of the adenoma -carcinoma sequence, suggesting a link to the APC/b-catenin/ TCF pathway (Wnt-signalling pathway). Therefore, the effect of the NSAID sulindac on nuclear (nonphosphorylated) b-catenin and b-catenin/TCF-mediated transcription was investigated. Nuclear b-catenin expression was assessed in pretreatment colorectal adenomas and in adenomas after treatment with sulindac from five patients with familial adenomatous polyposis (FAP). Also, the effect of sulindac sulphide on b-catenin/TCF-mediated transcription was studied. Adenomas of FAP patients collected after treatment with sulindac for up to 6 months showed less nuclear b-catenin expression compared to pretreatment adenomas of the same patients. Sulindac sulphide abrogated b-catenin/TCF-mediated transcription in the CRC cell lines DLD1 and SW480, and decreased the levels of nonphosphorylated b-catenin. As a result, the protein levels of the positively regulated TCF targets Met and cyclin D1 were downregulated after sulindac treatment. This study provides in vivo and in vitro evidence that nuclear b-catenin localisation and b-catenin/TCF-regulated transcription of target genes can be inhibited by sulindac. The inhibition of Wnt-signalling provides an explanation for the COX-2-independent mechanism of chemoprevention by NSAIDs. . Also, in patients with familial adenomatous polyposis (FAP), an autosomal dominantly inherited disorder characterised by the development of numerous colorectal adenomas at a young age, the NSAIDs sulindac and indomethacin can cause regression of adenomas (Giardiello et al, 1993;Nugent et al, 1993;Spagnesi et al, 1994;Hirota et al, 1996;Winde et al, 1997;Picariello et al, 1998). The chemopreventive effect of NSAIDs appears mediated by the induction of apoptosis and cell cycle arrest (Pasricha et al, 1995;DuBois and Smalley, 1996;Piazza et al, 1997;Keller et al, 1999;Shiff and Rigas, 1999). The molecular mechanisms underlying these biological effects are not completely understood. Nonsteroidal anti-inflammatory drugs inhibit the enzymatic activity of cyclooxygenase (COX)-1 and -2, enzymes that convert arachidonic acid into prostaglandins (Shiff and Rigas, 1999). However, COX-independent mechanisms may also play a role, since NSAIDs inhibit the growth of colon cancer cell lines lacking COX-2 expression (Hanif et al, 1996;Zhang et al, 1999;Smith et al, 2000).Oncogenic activation of the Wnt-signalling pathway by mutations in Adenomatous polyposis coli (APC) or b-catenin, which results in the accumulation and nuclear translocation of b-catenin and in b-catenin/TCF4-regulated transcription of TCF target genes, is mandatory for the initial neoplastic transformation of intestinal epithelium (reviewed in Kinzler and Vogelstein, 1996...

CADASIL is an autosomal dominant inherited disease, characterized by mid-adult onset of cerebrovascular disease and dementia. CADASIL is caused by mutations in the NOTCH3 gene, which encodes the NOTCH3 protein. Pathogenic mutations in CADASIL are highly distinctive in the sense that they lead to the loss or gain of a cysteine residue in 1 of the 34 EGFr domains of the NOTCH3 protein. The majority are missense mutations, but small deletions, insertions and splice-site mutations have been reported, which typically also lead to a numerical cysteine alteration. Whether numerical cysteine-altering mutations are a rule in CADASIL remains subject of debate, as there are reports suggesting pathogenicity of other types of mutations. However, for most of these the association with CADASIL was later revoked or is questionable. Here, we discuss and provide recommendations for the interpretation of NOTCH3 mutations in the diagnosis of CADASIL.

Pbx2 is one of four mammalian genes that encode closely related TALE homeodomain proteins, which serve as DNA binding partners for a subset of Hox transcription factors. The expression and contributions of Pbx2 to mammalian development remain undefined, in contrast to the essential roles recently established for family members Pbx1 and Pbx3. Here we report that Pbx2 is widely expressed during embryonic development, particularly in neural and epithelial tissues during late gestation. Despite wide Pbx2 expression, mice homozygous mutant for Pbx2 are born at the expected Mendelian frequencies and exhibit no detectable abnormalities in development and organogenesis or reduction of long-term survival. The lack of an apparent phenotype in Pbx2 ؊ / ؊ mice likely reflects functional redundancy, since the Pbx2 protein is present at considerably lower levels than comparable isoforms of Pbx1 and/or Pbx3 in embryonic tissues. In postnatal bone marrow and thymus, however, Pbx2 is the predominant high-molecular-weight (MW)-isoform Pbx protein detectable by immunoblotting. Nevertheless, the absence of Pbx2 has no measurable effect on steady-state hematopoiesis or immune function in adult mice, suggesting possible compensation by low-MW-isoform Pbx proteins present in these tissues. We conclude that the roles of Pbx2 in murine embryonic development, organogenesis, hematopoiesis, immune responses, and long-term survival are not essential.

Background: Episodic ataxia (EA) is variably associated with additional neurologic symptoms. At least 4 genes have been implicated. Recently, a mutation in the SLC1A3 gene encoding the glutamate transporter EAAT1 was identified in a patient with severe episodic and progressive ataxia, seizures, alternating hemiplegia, and migraine headache. The mutant EAAT1 showed severely reduced uptake of glutamate. The syndrome was designated EA6 and shares overlapping clinical features with EA2, which is caused by mutations in CACNA1A. Objective: To test the role of the SLC1A3 gene in EA. Design: Genetic and functional studies. We analyzed the coding region of the SLC1A3 gene by direct sequencing. Setting: Academic research. Patients: DNA samples from 20 patients with EA (with or without interictal nystagmus) negative for CACNA1A mutations were analyzed. Main Outcome Measures: We identified 1 novel EAAT1 mutation in a family with EA and studied the functional consequences of this mutation using glutamate uptake assay. Results: We identified a missense C186S mutation that segregated with EA in 3 family members. The mutant EAAT1 showed a modest but significant reduction of glutamate uptake. Conclusions: We broadened the clinical spectrum associated with SLC1A3 mutations to include milder manifestations of EA without seizures or alternating hemiplegia. The severity of EA6 symptoms appears to be correlated with the extent of glutamate transporter dysfunction.

Activity of receptor tyrosine kinases (RTK) in colorectal cancer (CRC) is associated with enhanced tumor growth and a poorer prognosis. In addition, cyclooxygenase-2 (COX-2) expression contributes to tumor growth and invasion. COX-2 inhibitors exhibit important anticarcinogenic potential against CRC, but the molecular mechanism underlying this effect and the relation with RTK signaling remain the subject of intense research effort. Therefore, the rapid effects of COX-2 inhibition in CRC on the complement of all cellular kinases were investigated using a kinase substrate peptide array, Western blotting, transfection, small interfering RNA assays, and CRC cell lines. The resulting alterations in the kinome profile revealed that celecoxib, a selective COX-2 inhibitor, impairs phosphorylation of substrates for the RTKs c-Met and insulinlike growth factor receptor, resulting in decreased downstream signaling. The decrease in c-Met activation is accompanied with an increase in glycogen synthase kinase 3B kinase activity together with a rapid increase in phosphorylation of B-catenin. In agreement, a significant reduction of B-catenin-T-cell factor-dependent transcription is observed both with celecoxib and selective inhibition of c-Met phosphorylation by small molecules. Hence, corepression of c-Met-related and Bcatenin-related oncogenic signal transduction seems a major effector of celecoxib in CRC, which provides a rationale to use c-Met inhibitors and celecoxib analogous to target c-Met and Wnt signaling in a therapeutic setting for patients with CRC.