Gene expression profiling to predict the risk of locoregional recurrence in breast cancer: a pooled analysis.

The 70-gene signature (MammaPrint) has been developed to predict the risk of distant metastases in breast cancer and select those patients who may benefit from adjuvant treatment. Given the strong association between locoregional and distant recurrence, we hypothesize that the 70-gene signature will also be able to predict the risk of locoregional recurrence (LRR). 1,053 breast cancer patients primarily treated with breast-conserving treatment or mastectomy at the Netherlands Cancer Institute between 1984 and 2006 were included. Adjuvant treatment consisted of radiotherapy, chemotherapy, and/or endocrine therapy as indicated by guidelines used at the time. All patients were included in various 70-gene signature validation studies. After a median follow-up of 8.96 years with 87 LRRs, patients with a high-risk 70-gene signature (n = 492) had an LRR risk of 12.6% (95% CI 9.7-15.8) at 10 years, compared to 6.1% (95% CI 4.1-8.5) for low-risk patients (n = 561; P < 0.001). Adjusting the 70-gene signature in a competing risk model for the clinicopathological factors such as age, tumour size, grade, hormone receptor status, LVI, axillary lymph node involvement, surgical treatment, endocrine treatment, and chemotherapy resulted in a multivariable HR of 1.73 (95% CI 1.02-2.93; P = 0.042). Adding the signature to the model based on clinicopathological factors improved the discrimination, albeit non-significantly [C-index through 10 years changed from 0.731 (95% CI 0.682-0.782) to 0.741 (95% CI 0.693-0.790)]. Calibration of the prognostic models was excellent. The 70-gene signature is an independent prognostic factor for LRR. A significantly lower local recurrence risk was seen in patients with a low-risk 70-gene signature compared to those with high-risk 70-gene signature.

Optimized outcome prediction in breast cancer by combining the 70-gene signature with clinical risk prediction algorithms.

Clinical guidelines for breast cancer treatment differ in their selection of patients at a high risk of recurrence who are eligible to receive adjuvant systemic treatment (AST). The 70-gene signature is a molecular tool to better guide AST decisions. The aim of this study was to evaluate whether adding the 70-gene signature to clinical risk prediction algorithms can optimize outcome prediction and consequently treatment decisions in early stage, node-negative breast cancer patients. A 70-gene signature was available for 427 patients participating in the RASTER study (cT1-3N0M0). Median follow-up was 61.6 months. Based on 5-year distant-recurrence free interval (DRFI) probabilities survival areas under the curve (AUC) were calculated and compared for risk estimations based on the six clinical risk prediction algorithms: Adjuvant! Online (AOL), Nottingham Prognostic Index (NPI), St. Gallen (2003), the Dutch National guidelines (CBO 2004 and NABON 2012), and PREDICT plus. Also, survival AUC were calculated after adding the 70-gene signature to these clinical risk estimations. Systemically untreated patients with a high clinical risk estimation but a low risk 70-gene signature had an excellent 5-year DRFI varying between 97.1 and 100 %, depending on the clinical risk prediction algorithms used in the comparison. The best risk estimation was obtained in this cohort by adding the 70-gene signature to CBO 2012 (AUC: 0.644) and PREDICT (AUC: 0.662). Clinical risk estimations by all clinical algorithms improved by adding the 70-gene signature. Patients with a low risk 70-gene signature have an excellent survival, independent of their clinical risk estimation. Adding the 70-gene signature to clinical risk prediction algorithms improves risk estimations and therefore might improve the identification of early stage node-negative breast cancer patients for whom AST has limited value. In this cohort, the PREDICT plus tool in combination with the 70-gene signature provided the best risk prediction.

Pharmacogenetics: from bench to byte--an update of guidelines.

Currently, there are very few guidelines linking the results of pharmacogenetic tests to specific therapeutic recommendations. Therefore, the Royal Dutch Association for the Advancement of Pharmacy established the Pharmacogenetics Working Group with the objective of developing pharmacogenetics-based therapeutic (dose) recommendations. After systematic review of the literature, recommendations were developed for 53 drugs associated with genes coding for CYP2D6, CYP2C19, CYP2C9, thiopurine-S-methyltransferase (TPMT), dihydropyrimidine dehydrogenase (DPD), vitamin K epoxide reductase (VKORC1), uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1), HLA-B44, HLA-B*5701, CYP3A5, and factor V Leiden (FVL).

The role of inflammation on atherosclerosis, intermediate and clinical cardiovascular endpoints in type 2 diabetes mellitus.

BACKGROUND: Type 2 diabetes mellitus (T2DM) is associated with increased cardiovascular morbidity and mortality. Sub-clinical systemic inflammation is often present in T2DM patients. Systemic inflammation has also been implicated in the pathophysiology of atherosclerosis. This review investigates the direct evidence present in literature for the effect of inflammation on atherosclerosis, specifically in the setting of T2DM. Special emphasis is given to the pathogenesis of atherosclerosis as well as intermediate and clinical cardiovascular endpoints. The important role of deteriorated endothelial function in T2DM was excluded from the analysis. METHODS: Extensive literature searches were performed using the PubMed and Web of Science databases. Articles were identified, retrieved and accepted or excluded based on predefined criteria. RESULTS: Substantial evidence was found for an important inflammatory component in the pathogenesis of atherosclerosis in T2DM, demonstrated by inflammatory changes in plaque characteristics and macrophage infiltration. Most epidemiologic studies found a correlation between inflammation markers and intermediate cardiovascular endpoints, especially intima-media thickness. Several, but not all clinical trials in T2DM found that reducing sub-clinical inflammation had a beneficial effect on intermediate endpoints. When regarding cardiovascular events however, current literature consistently indicates a strong relationship between inflammation and clinical endpoints in subjects with T2DM. CONCLUSION: Current literature provides direct evidence for a contribution of inflammatory responses to the pathogenesis of atherosclerosis in T2DM. The most consistent relation was observed between inflammation and clinical endpoints.

The Hormone Domain of the Vasopressin Prohormone is Required for the Correct Prohormone Trafficking Through the Secretory Pathway.

It has long been known that under intracellular conditions vasopressin associates tightly to neurophysin, which is present in the same prohormone. As the association has been suggested to play a role during hormone biosynthesis, its role was studied in a cellular context by expressing mutant vasopressin precursors in Neuro2A cells. Mutant vasopressin precursors, in which the association between the vasopressin and neurophysin domains was prevented either by deleting the vasopressin domain from the precursor or by substitution of the essential Tyr2 residue in vasopressin for Gly, were neither processed nor targeted into secretory granules. Rather, both provasopressin mutants were retained in the endoplasmic reticulum. Our results demonstrate that the vasopressin domain is crucial for correct trafficking of the prohormone through the secretory pathway, and suggest that vasopressin-neurophysin association provides correct prohormone folding in the endoplasmic reticulum.

Neonatal life support during magnetic resonance imaging.

Magnetic resonance techniques are required frequently for the assessment of the brain of ill neonates. In the present study, the effects of a 1.5 T MR scanner on devices for life support were assessed. A ventilator (Dräger Babylog 2000) was tested in the 1.5 T magnet, using a neonatal ventilation tester and 1.5-5 m tubes. In a special MR incubator, temperature and humidity were measured at 1-min intervals. Infusion was tested with the pump outside the magnet room: infusion rates and time to alarm were tested with 7-m tubes. The ventilator performed normally at a magnetic field line of 2 mT, although the alarms failed. The incubator created a temperature of 35.9 degrees C and humidity of 40.7%, which was acceptable for examinations of 45 min. The alarm limits of the infusion pump placed outside the magnet at 7 m were within company limits. The study indicates that magnetic resonance examinations can be performed safely in ill preterm neonates who require life-support devices.

Familial neurohypophysial diabetes insipidus in a large Dutch kindred: effect of the onset of diabetes on growth in children and cell biological defects of the mutant vasopressin prohormone.

Familial neurohypophysial diabetes insipidus (FNDI) is an autosomal dominant trait in which expression of a mutant vasopressin prohormone reduces vasopressin production. We investigated the NP85 Cys-->Gly mutant vasopressin prohormone in a large kindred in The Netherlands. We demonstrate that growth retardation is an important early sign in two children from this kindred, which recuperates by substitution therapy with 1-desamino-8-D-arginine vasopressin. To obtain clues about the basis for the dominant inheritance of FNDI, we analyzed the trafficking and processing of the mutant vasopressin prohormone in cell lines by metabolic labeling and immunoprecipitation. The mutant vasopressin prohormone was retained in the endoplasmic reticulum and thus was not processed to vasopressin. This defect was not caused by dimerization of the vasopressin prohormone via its unpaired cysteine residue. High level expression of the mutant vasopressin prohormone in cell lines resulted in strong accumulation in the endoplasmic reticulum and an altered morphology of this organelle. We hypothesize that disturbance of the endoplasmic reticulum results in dysfunction and ultimately cell death of the cells expressing the mutant prohormone. Our data support the hypothesis that FNDI is a progressive neurodegenerative disease with delayed onset of symptoms. Its treatment requires early detection of symptoms for which growth parameters are useful.

A diabetes insipidus vasopressin prohormone altered outside the central core of neurophysin accumulates in the endoplasmic reticulum.

Over 20 mutations affecting the neurophysin moiety of the vasopressin prohormone, have been identified in families suffering from familial neurohypophysial diabetes insipidus (FNDI). Only one of these, NP87E-->stop, is located outside the central conserved domain implicated in sorting of the vasopressin prohormone. To obtain clues about the mechanism of induction of FNDI by this atypical mutant we stably expressed wild type and NP87E-->stop vasopressin prohormones in (neuro)endocrine cell lines. Metabolic labeling and immunoprecipitation demonstrated reduced processing of the mutant prohormone to neurophysin. In addition, evoked secretion of neurophysin and vasopressin was diminished, suggesting that part of the mutant is retained in another intracellular compartment than the secretory granules. Indeed, immunofluorescence demonstrated accumulation of the truncated vasopressin prohormone in the endoplasmic reticulum. We conclude that the presence of the vasopressin moiety and the central conserved core of the neurophysin domain suffices for sorting and processing, but not for efficient endoplasmic reticulum exit of the vasopressin-neurophysin molecule.

Processing of frameshifted vasopressin precursors.

Biosynthesis of the vasopressin (VP) prohormone in magnocellular neurones of the hypothalamo-neurohypophysial system comprises endoplasmic reticulum (ER) transit, sorting into the regulated secretory pathway and subsequent processing in the individual proteins VP, neurophysin and a glycoprotein. These processes are severely disrupted in the homozygous diabetes insipidus (di/di) Brattleboro rat, which expresses a mutant VP precursor due to a single nucleotide deletion in the neurophysin region of the VP gene resulting in VP deficiency. Previous studies have shown the presence of additional frameshift mutations in VP transcripts, in solitary magnocellular neurones of the di/di rat due to a GA dinucleotide deletion resulting in two different mutant VP precursors with partly restored reading frame. Frameshifted VP precursors are also expressed in several magnocellular neurones in wild-type rats. In this study, we determined if the +1 frameshifted precursors from di/di and wild-type rats can lead to biosynthesis of the hormone VP. Therefore, eukaryotic expression plasmids containing the frameshifted VP cDNAs were transiently expressed in peptidergic tumour cell lines, and cells were analysed by reversed phase high-performance liquid chromatography and specific radioimmunoassays, and by immunofluoresence. Neuro2A neuroblastoma cells expressing the +1 frameshifted precursors of di/di rats retained products in the cell body. Only precursor or insignificant quantities of neurophysin-immunoreactive products were detected. In contrast, in AtT20 cells, frameshifted VP precursors were at least partly processed to yield the VP peptide, indicating that they have access to the regulated secretory pathway. Comparison between the two cell lines showed a very slow ER transit of the wild-type prohormone combined with inefficient processing in Neuro2A cells. The results show that mutant precursors can reach the regulated secretory pathway if ER transport is sufficiently rapid as in the case of AtT20 cells. This suggests that the di/di rat may regain the capacity to biosynthesize authentic VP through these +1 frameshifted precursors in magnocellular neurones.

Two type XVII collagen (BP180) mRNA transcripts in human keratinocytes: a long and a short form.

We have analysed BP180 mRNA expression in normal human keratinocytes. Here we report the presence in normal keratinocytes of two COL17A1 transcripts which differ by 0.6 kb in length. Both mRNAs hybridized on Northern blot with probes directed to sequences encoding intracellular and extracellular fragments of BP180. By BLAST homology search alignments we extended the 3' untranslated region (3'UTR) of the known BP180 mRNA sequence by 877 bases to completion. Three of 20 cDNAs identified by BLAST searches contained a 610 bp deletion in this new 3'UTR sequence. Northern blot analysis with a probe complementary to this deleted sequence showed binding only to the larger mRNA. The deletion of 610 nucleotides in the smaller mRNA was verified by reverse transcription-PCR and sequencing. Genomic PCR showed the new sequence to be an extension of exon 56 of the COL17A1 gene which suggests that the second mRNA is generated by differential splicing. In normal keratinocytes the level of the smaller transcript was 5-15% of that of the larger transcript whereas in a squamous cell carcinoma cell line this ratio was reversed, the smaller mRNA being three times more abundant than the larger mRNA. The biological significance of this newly identified transcript in protein synthesis and tissue expression or in cell differentiation, proliferation or adhesion is as yet unknown.

Mutations in the vasopressin prohormone involved in diabetes insipidus impair endoplasmic reticulum export but not sorting.

Familial neurohypophysial diabetes insipidus is characterized by vasopressin deficiency caused by heterozygous expression of a mutated vasopressin prohormone gene. To elucidate the mechanism of this disease, we stably expressed five vasopressin prohormones with a mutation in the neurophysin moiety (NP14G-->R, NP47E-->G, NP47DeltaE, NP57G-->S, and NP65G-->V) in the neuroendocrine cell lines Neuro-2A and PC12/PC2. Metabolic labeling demonstrated that processing and secretion of all five mutants was impaired, albeit to different extents (NP65G-->V >/= NP14G-->R > NP47DeltaE >/= NP47E-->G > NP57G-->S). Persisting endoglycosidase H sensitivity revealed these defects to be due to retention of mutant prohormone in the endoplasmic reticulum. Mutant prohormones that partially passed the endoplasmic reticulum were normally targeted to the regulated secretory pathway. Surprisingly, this also included mutants with mutations in residues involved in binding of vasopressin to neurophysin, a process implicated in targeting of the prohormone. To mimick the high expression in vasopressin-producing neurons, mutant vasopressin prohormones were transiently expressed in Neuro-2A cells. Immunofluorescence displayed formation of large accumulations of mutant prohormone in the endoplasmic reticulum, accompanied by redistribution of an endoplasmic reticulum marker. Our data suggest that prolonged perturbation of the endoplasmic reticulum eventually leads to degeneration of neurons expressing mutant vasopressin prohormones, explaining the dominant nature of the disease.

Type XVII collagen (BP180) and LAD-1 are present as separate trimeric complexes.

This study characterized the high molecular mass BP180 complex that is observed when unheated sodium dodecyl sulfate extracts of human skin or keratinocytes are subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. In heated extracts BP180 is present as a monomer with a molecular weight of 180 kDa, in unheated extracts BP180 runs at a molecular weight position over 500 kDa. By preincubating the unheated extracts at temperatures between 31 degrees C and 40 degrees C, the high molecular weight complex could be "melted" down to monomeric BP180. Under the conditions employed the T1/2 of the dissociation process was between 35 degrees C and 36 degrees C. The temperature resistance of the high molecular weight complex was used to analyze its molecular composition by performing two-dimensional electrophoresis with a "low-temperature" first dimension step and a "high-temperature" second dimension step. Silver staining and immunoblotting of the two-dimensional gels revealed the high molecular weight complex to be composed of solely BP180, indicating that the complex is the nondissociated homotrimeric form of BP180. The 120 kDa linear IgA dermatosis antigen (LAD-1) is an collagenous anchoring filament protein with homology to the extracellular collagenous part of BP180. Two-dimensional immunoblotting showed that LAD-1, as BP180, is also present as a high molecular mass complex and does not form mixed complexes with BP180.

Hypothyroidism in the rat results in decreased soleus motoneurone soma size.

Adult female rats were thyroidectomized. After an average of 17 weeks, horseradish peroxidase (HRP) was injected into the right side soleus muscle. Two days later, left side soleus muscle properties were recorded and muscles and spinal cord were removed for further histological measurements. Soleus muscles from hypothyroid rats no longer contained type IIA fibers. Contraction and half-relaxation times of twitches had increased significantly compared to control rats. The average cross-sectional surface areas of HRP-labeled soleus motoneurones from hypothyroid rats were slightly but significantly smaller than those of control rats. A similar decrease in size was found for other (presumed moto-) neurones lying ventrolaterally to the soleus motor nucleus. It is concluded that changes in the soleus muscle fiber composition, as caused by lowered levels of thyroid hormone, are paralleled by corresponding changes in the size of its motoneurones and also of other spinal (moto)neurones.

The endoplasmic reticulum-resident stress protein gp96 binds peptides translocated by TAP.

The endoplasmic reticulum (ER)-resident stress protein gp96 induces a major histocompatibility complex class I-restricted cytotoxic T lymphocyte (CTL) response against antigens present in the cells from which it has been prepared. In this study, photoreactive peptides were translocated into the ER by the transporter associated with antigen processing (TAP). These peptides can be cross-linked specifically to gp96. Thus, we provide the first evidence that gp96 binds TAP-translocated peptides which have been implicated in the induction of specific CTL responses after immunization with gp96 (Srivastava, P. K. et al., Immunogenetics 1994. 39: 93).

Revertant mosaicism in epidermolysis bullosa caused by mitotic gene conversion.

Mitotic gene conversion acting as reverse mutation has not been previously demonstrated in human. We report here that the revertant mosaicism of a compound heterozygous proband with an autosomal recessive genodermatosis, generalized atrophic benign epidermolysis bullosa, is caused by mitotic gene conversion of one of the two mutated COL17A1 alleles. Specifically, the maternal allele surrounding the mutation site on COL17A1 (1706delA) showed reversion of the mutation and loss of heterozygosity along a tract of at least 381 bp in revertant keratinocytes derived from clinically unaffected skin patches; the paternal mutation (R1226X) remained present in all cell samples. Revertant mosaicism represents a way of natural gene therapy.

Multiple regions of the transporter associated with antigen processing (TAP) contribute to its peptide binding site.

The transporter associated with Ag processing (TAP) translocates cytosolic peptides into the endoplasmic reticulum, where they can bind to MHC class I molecules. TAP does not translocate all peptides with equal efficiency, but selects peptides with regard to both their length and their sequence and, in this manner, affects the pool of peptides available for binding to MHC class I molecules. It has been demonstrated that peptide selection by TAP predominantly occurs during the first step in the translocation process, namely the association of the peptide with a binding site present on the TAP molecule. In this study, we identify four regions, two on the TAP1 and two on the TAP2 subunit, that make major contributions to this binding site. For both TAP1 and TAP2, the identified regions overlap with the cytosol-membrane boundaries of the two transmembrane segments closest to the ATP binding site. Our data are consistent with a model in which the transmembrane segments of TAP form a pore in the membrane, with the peptide binding site being formed by the cytosolic mouth of this pore.

A point mutation in the human transporter associated with antigen processing (TAP2) alters the peptide transport specificity.

The heterodimeric transporter associated with antigen processing (TAP1/TAP2) translocates peptides from the cytosol into the endoplasmic reticulum where loading of major histocompatibility complex class I molecules takes place. TAP transporters from different species are known to exhibit distinct transport specificities with regard to the C-terminal amino acid (aa) of peptides. Thus, human TAP (hTAP), and rat TAP (rTAP) containing the rTAP2a allele are rather promiscuous, whereas mouse TAP (mTAP), and rTAP containing the rTAP2a allele are restrictive and select against peptides with C-terminal small polar/hydrophobic or positively charged aa. The structural basis for this selectivity is not clear. To assess the relative contribution of the TAP1 and TAP2 subunits to transport specificity, we have constructed and analyzed interspecies TAP hybrids and point mutants of hTAP2 expressed in Sf9 insect cells and in TAP-deficient T2 cells. Transport assays with 20 C-terminal variants of the peptide RYWANATRSX showed that: first, transport specificity with regard to C-terminal aa is mainly influenced by TAP2, but TAP1 can also contribute. Second, the selective transport of peptides with C-terminal positively charged aa is critically controlled by the amino-terminal region (1-361) on the TAP2 chain, while transport of peptides with C-terminal small polar/hydrophobic aa is determined by residues located within as well as outside the region 1-361. Third, a single point mutation in hTAP2 (374A-->D) resulted in a drastic alteration of the transport pattern. These results indicate that both TAP1 and TAP2 contribute to efficient peptide transport and that single point mutations in hTAP2 are able to alter the peptide transport specificity. This opens the possibility that naturally occurring mutations in one of the hTAP subunits may alter epitope selection in vivo.

Identification of a contact region for peptide on the TAP1 chain of the transporter associated with antigen processing.

The transporter associated with Ag processing (TAP) translocates cytosolic peptides into the endoplasmic reticulum for presentation by MHC class 1 molecules. Recently, the actual peptide translocation step has been suggested to be preceded by binding of the peptide to TAP. In this study, we investigated the peptide binding site of TAP and its relevance for peptide selection by cross-linking of translocatable peptides. Our data demonstrate, first, that for a TAP heterodimer containing the rat TAPu allelic product, which selects peptides on basis of their C terminus, the translocation efficiency correlates with the peptide binding efficiency. Second, peptides having the cross-linker at different positions all label both the TAP1 and the TAP2 subunit after binding to the heterodimer, indicating that both TAP subunits contribute directly to the peptide binding site and contact most or all amino acids of a bound peptide. Third, by enzymatic digestion and the use specific antisera, we identified a domain of human TAP1 that contributes to the peptide binding site. This domain contains the two hydrophobic and thus putative transmembrane regions closest to the ATP binding sites. We conclude that the peptide binding site controls the selectivity of TAP and is composed of domains of both TAP1 and TAP2, which each contact the bound peptide over all or most of its length. Moreover, the major contact site(s) for peptide on TAP1 are located within or close to the two putative transmembrane regions adjacent to the ATP binding site.

TAP polymorphism does not influence transport of peptide variants in mice and humans.

The major histocompatibility complex (MHC)-encoded transporter associated with antigen processing (TAP) delivers cytosolic peptides to the lumen of the endoplasmic reticulum (ER) for presentation by MHC class I molecules. For the rat, it has been demonstrated that TAP polymorphism results in the selection of different sets of peptides, the nature of the C terminus being of particular importance. Here, we investigated whether TAP polymorphism in mice and humans has functional consequences for transport of peptide sets variable at the C-terminal residues. Using cell lines of H-2d, H-2k, and H-2dxk haplotype and a panel of human lymphoblastoid cell lines expressing eight different TAP alleles, we detected species-specific transport patterns, but no significant influence of TAP polymorphism on peptide selection. In addition, peptides with different core sequences were translocated to the same extent by different TAP. These results suggest that a major contribution of human TAP polymorphism to disease progression and autoimmunity is not very likely.