The treatment with parenteral cefotriaxone followed by oral cotri

The treatment with parenteral cefotriaxone followed by oral cotrimoxazole is reserved to the forms with involvement of CNS for the ability of these antibiotics to overcome the blood-brain moreover barrier. Figure 1 Wipple��s disease: obstruction and perforated bowel lesions, characterized by edema and lymphadenopathy of intestinal wall. Figure 2 Wipple��s disease: lesions of mesentery with lipodystrophy. Figure 3 Wipple��s disease: intestinal mucosa biopsy with PAS-positive stain.
Smoking in pregnancy is a significant public health problem. In the United Kingdom, a country with strong tobacco control culture, a survey in 2011 found that 26% of pregnant women smoked at some point before or during pregnancy and 12% smoked constantly throughout gestation (Eastwood, 2011).

As smoking is a completely preventable cause of poor health outcomes for mothers and their babies, stopping smoking before or during pregnancy is vital. Unfortunately, though, there are few evidence-based cessation interventions that are proven to work for cessation in pregnancy. A systematic review investigating the predictors of quit attempts made by nonpregnant smokers, found that a lower number of previous quit attempts and higher levels of nicotine dependence were both inversely associated with cessation (Vangeli, Stapleton, Smit, Borland, & West, 2011). Factors that have been associated with increased number of quit attempts in pregnancy also include age and smoking duration (Yu, Park, & Schwalberg, 2002).

However, a recent systematic review found that having a partner who smoked, multiparity and increasing nicotine dependence had, in many studies, been found inversely associated with likelihood of achieving cessation (Schneider, Huy, Schutz, & Diehl, 2010). Additionally, socioeconomic factors such as increased income and educational levels of the mother and partner have also been shown to be associated with cessation in pregnancy (Ebert & Fahy, 2007; Mohsin & Bauman, 2005; Schneider et al., 2010), but these associations may be due to decline in smoking rates, which has been found to be lower in women from lower socioeconomic groups (US DHHS, 2004). Data from surveys conducted in the United Kingdom and Spain have also found that pregnant women with lower educational and socioeconomic levels have lower chances of cessation, whereas women who smoked fewer cigarettes, started smoking at an older age, had a partner who did not smoke or were primiparous were more likely to quit (Torrent et al., 2004). There is less evidence, though, about which factors might influence women��s success when using nicotine replacement therapy (NRT) in cessation attempts made during Cilengitide pregnancy.

Progress in the knowledge of hepatic anatomy is proportionally re

Progress in the knowledge of hepatic anatomy is proportionally related to changes in the evolution of liver transplant techniques. The anatomical segmentation that was defined by Couinaud has guided procedural conduct regarding the procedures for hepatic resection since 1957 (1, 2). The anatomical importance of the intrahepatic biliary ducts came up again in the late 1980s due sellckchem to the advent of liver transplantation from living donors (2, 3). Furthermore, the technique of Split-Liver transplantation allows a single organ from a cadaveric donor to be shared by two living recipients if one is a pediatric recipient while the other is an adult. In both of them the need exists for resection of the parenchyma and performance of multiple anastomoses of intrahepatic bile ducts. Taketomi et al.

reported a 4.1% incidence of postoperative biliary complications in donors (5). An understanding of the anatomy of the biliary tree and its main variations is essential in reduce the rate of biliary leakage and lessen morbidity related to the procedure (6, 7). In this context, increased safety may be provided by following the simple rule that the fewer anastomoses there are, the fewer complications there will be. Thus the determination of the points of confluence of the drainage ducts of each segment is of utmost importance. Despite the emphasized importance in liver transplantation, studying the anatomy of the biliary duct is essential in any surgery involving this organ. Many authors advocate the routine performance of retrograde cholangiopancreatography before any hepatic resection (8).

This statement gains strength when donors are still living, where more severe anomalies may also contraindicate the procedure. Methodology The objective of this study was to evaluate the anatomy of the biliary drainage of the left lobe of the liver through relevant parameters in hepatic resection. These parameters are specified above: Define relationships and distances between the confluence of the main bile ducts involved in the drainage of the left lobe, also noting its relationship with ligamentum venosum of the liver. Evaluate the confluence of the right hepatic duct (RHD) and the left hepatic duct (LHD), and the anomalies thereof. Evaluate the patterns of drainage of the duct of segment I (DSI) and the duct of segment IV (DSIV).

This study evaluated Brefeldin_A cadaveric human livers extracted within a period of up to 24 hours after death. The specimens were obtained from the Department of Forensic Medicine of Porto Alegre – RS. Approval was obtained from the Research and Ethics Committee prior to the beginning of the study. Forty-five organs were used, all of which were removed, during the autopsy while preserving the hiliary vascular pedicle, from the inferior vena cava above the renal veins and below the right atrium.

Blood samples from 36 donors were collected

Blood samples from 36 donors were collected then after signature of written informed consent, from June 2010 to October 2011. Twenty six of these samples were collected from NPC patients admitted to the Institut de Canc��rologie Gustave Roussy or Paris hospitals working in collaboration with this Institute (Table1). All these patients had evidence of active disease, either as a first occurrence or as a recurrence. Blood samples were generally collected before initiation of the treatment (Table1). Control samples were obtained from 9 consecutive patients admitted to the Department of Head and Neck Oncology at the Institut de Canc��rologie Gustave Roussy for non-NPC tumors. All these tumors were squamous cell carcinomas of the upper aero-digestive track. More clinical details are provided in Table2.

One additional control plasma sample was obtained from a healthy EBV-carrier donor. For all donors, plasma was separated from blood collected in EDTA tubes by centrifugation at 1700 g at 20��C for 15 min, aliquoted and frozen at �C 80��C. In virtually all cases, plasma separation and freezing was done in less than 2 hours following blood collection. Clinical staging and pathological diagnosis Tumor staging was performed according to UICC 7th edition (Union for International Cancer control) guidelines [24]. Histological classification was performed according to the 2005 WHO classification [25]. Except for 4 patients, the histological diagnosis was confirmed by detection of EBV products on tissue sections, either the LMP1 protein detected by immunohistochemistry (3 cases) or the EBERs (Epstein-Barr encoded RNAs) detected by in situ hybridization using commercial kits, mainly from Ventana Medical System (Illkirch, France) [26].

Isolation and chemical analysis of plasma lipoprotein fractions Plasma lipoproteins were isolated from 2 selected fasting plasma samples (2 to 3 mL) by density gradient ultracentrifugation in a Beckman SW41 Ti rotor at 40 000 rpm for 42 hours in a Beckman XL70 at 15��C as previously described [9]. Plasma density was increased to d=1.21 g/mL by addition of dry, solid KBr. A discontinuous density gradient was constructed as follows: 2 mL of NaCl-KBr solution of d=1.24 g/mL; 3 mL of plasma adjusted to a background density of d=1.21 g/mL; 2 mL of d=1.063 g/mL; 2.5 mL of d=1.019 g/mL; and finally, 2.5 mL of NaCl solution of d=1.006 g/mL.

After centrifugation, gradients were collected from the top of the tubes with an Eppendorf precision pipette in 30 fractions corresponding to VLDL (density <1.006 g/mL, fraction number 1), IDL (density from 1.006 g/mL to 1.019 g/mL, fraction number 2), GSK-3 10 LDL subfractions (density from 1.019 g/mL to 1.063 g/mL, fraction number 3 to 12) and 11 HDL subfractions (density from 1.063 g/mL to 1.179 g/mL, fraction number 13 to 23).

Figure 3 Trypsin inhibitors increase the amount of trypsin needed

Figure 3 Trypsin inhibitors increase the amount of trypsin needed to potentiate fibrocyte potentiation. Albumin is Necessary for Trypsin to Potentiate Fibrocyte Differentiation selleck compound To test the hypothesis that trypsin acts on a protein supplement in the media to potentiate fibrocyte differentiation, we removed the protein supplements from our defined medium and added trypsin to this protein-free media. According to the manufacturer, Fibrolife medium is protein-free. Trypsin added to Fibrolife media lacking all three protein supplements (albumin, insulin and transferrin) did not potentiate fibrocyte differentiation (Figure 4A). At concentrations of 5 ��g/ml and higher, trypsin significantly decreased both fibrocyte numbers and the number of adhered cells (Figures 4A and 4B), presumably by decreasing cell adhesion.

These results suggest that trypsin acts on a protein supplement to indirectly potentiate fibrocyte differentiation, or that a protein supplement is necessary for trypsin��s potentiation to occur. Figure 4 Trypsin does not potentiate fibrocyte differentiation in medium lacking protein supplements. Serum-free media contains three proteins: insulin, transferrin, and albumin. To determine whether insulin, transferrin or albumin potentiates fibrocyte differentiation when exposed to trypsin, we purified human and bovine albumin and made media containing only insulin, transferrin, or albumin. When TPCK-treated trypsin-coated agarose beads were used to trypsinize culture media containing purified human or bovine albumin, the trypsinized media potentiated fibrocyte formation following the removal of the beads and addition to PBMC (Figure 5A).

Fibrocyte potentiation did not occur after the addition of protein-free, insulin-containing, or transferrin-containing media trypsinized in the same fashion (Figure 5A). To verify that TPCK did not influence fibrocyte differentiation, we digested human-albumin containing SFM with non TPCK-treated trypsin beads. Media containing human albumin digested by non TPCK-treated trypsin-beads also potentiated fibrocyte differentiation (Figure 5B). Using trypsin-coated beads to directly digest bovine and human albumin into fragments, and then adding those fragments to protein-free medium also potentiated fibrocyte differentiation compared to undigested controls (Figure 6A).

SDS-PAGE gels indicated that the protease treatment of albumin caused the formation of digestion products (Figure 6B). These results suggest that a trypsin fragment of albumin may potentiate fibrocyte differentiation. Figure 5 Serum-free medium containing albumin potentiates fibrocyte differentiation after temporary mixing with Cilengitide trypsin-coated agarose beads. Figure 6 Albumin potentiates fibrocyte differentiation after temporary mixing with trypsin-coated agarose beads.

Furthermore, hK1 levels were not associated with the cellular typ

Furthermore, hK1 levels were not associated with the cellular type and indexes of aggressiveness. An association was instead found with tumour location, with the highest values in GISTs of the small intestine. Our in vitro data show that expression levels in the GIST cell line are increased by hypoxia/starvation, pointing to the possibility that areas of the tumour far away from inhibitor Carfilzomib the vasculature could be stimulated to produce larger amounts of hK1. This is, at least in part, in line with the prevalent localization of hK1 in the central avascular core of GIST882 xenografts. In vitro studies on two different cell lines derived from Imatinib-resistant or Imatinib-sensitive tumours showed the common expression of hK1.

Cell biology results suggest two distinct molecular mechanisms by which hK1 could be implicated in GIST growth: (1) invasion through degradation of the ECM and (2) induction/stabilisation of host-derived tumour vasculature. A direct action of hK1 as an ECM-degrading enzyme could be complemented by the ability of hK1 to cleave and activate other proteases, such as pro-MMPs (Menashi et al, 1994). The proinvasive action was verified by a gene titration approach, using siRNA to decrease native hK1 expression and Ad.hK1 to force hK1 production. Silencing resulted in inhibition of GIST invasive capacity and overexpression enhanced it. The effect of silencing was further confirmed using inhibitors of hK1 activity. It is therefore likely that changes in hK1 production, release and clearance by endogenous inhibitors may confer a different invasive profile to the tumour.

Coculture experiments showed that GIST882 cells exert an attractive action on HUVEC, which is at least in part due to activation of kinin receptors, as verified by the use of receptor antagonists. Furthermore, GIST882-conditioned medium stimulates HUVEC to form more robust branches than those observed using unconditioned medium. The fact that kallistatin, which rapidly binds hK1 and inhibits its activity in vitro (Zhou et al, 1992), blocks the strengthening action of GIST882 cells on HUVEC networks argues in favour of hK1 as a promoter of cancer angiogenesis. Kallistatin itself was previously identified as an inhibitor of angiogenesis in gastric carcinomas (Zhu et al, 2007).

In the retrospective analysis of human GISTs, we could not find any correlation between hK1 expression and vascular density, suggesting Batimastat that other mechanisms may overwhelm or confound the proangiogenic effect of hK1 in vivo. On the other side, although HUVECs are widely used as models for tumoural angiogenesis, different mechanisms might drive cancer endothelial cells. In conclusion, results of our study show for the first time that hK1 is implicated in GIST invasion and angiogenesis. GIST882 xenografts express and release hK1 into the circulation, a result that calls for further validation of hK1 as a potential diagnostic biomarker.

Since then, legal advertising in Australia has been largely limit

Since then, legal advertising in Australia has been largely limited to point of sale displays (for which statewide restrictions were introduced between 1997 and 2006) and sponsorship though of international sporting events with exemptions from the bans, such as Grand Prix car racing, which continued to display tobacco industry sponsors�� logos until 2006 and contributed greatly to awareness of the Marlboro brand in particular (Scollo & Winstanley, 2008). The Australian menthol market is split between several ��stand-alone�� menthol brands and menthol ��line extensions�� within ��brand families�� where the original or ��parent�� brand is nonmenthol. One ��stand-alone�� menthol brand, Philip Morris�� Alpine (since line extended into an all-menthol brand family), has long been the most well-recognized menthol brand.

Advertising for Alpine prior to the end of electronic and print media advertising was highly feminized and strongly focused on younger smokers (Carter, 2001, 2003a, 2003b). There are also suggestions that marketing efforts were directed toward adolescent smokers. From 1986 to 1989, Alpine was one of a handful of Australian brands to be sold in packs of 15 cigarettes. This pack size was likely to have a strong appeal to adolescents due to its cheaper price. Advertising also focused on the small size of the pack, suggesting how easily it could be hidden. In more recent years, Philip Morris used various forms of ��stealth marketing��, including fashion events to promote the Alpine brand to young women (Carter, 2001, 2003a, 2003b; Harper, 2001; Winstanley et al., 1995).

Internal Philip Morris documents show that the company was concerned about the effect of increasing advertising restrictions on ��image-driven�� brands and that maintaining the market share of Alpine among younger women smokers was a high priority for the company (Philip Morris, 1987; 1994a; 1994b). One document (Philip Morris, 1994b), concerning a proposal to redesign the package, noted that Alpine retained a strong share of new smokers (around twice that of its total smoker share) but that its declining share of the ��key�� 18�C24 segment posed a threat to the long-term future of the GSK-3 brand. In this paper, we use data from three national prevalence studies in Australia to examine trends in the market share of menthol cigarettes (Alpine among adolescents) over the period 1980�C2008. We also explore whether the trends are consistent with Alpine being a starter cigarette for Australian adolescents. Methods Data Sources Adolescent Smoking The adolescent data are taken from the triennial Australian Secondary Students Alcohol and Drug Survey (ASSAD).

Their median age was 57 years, and 42 of 45 patients (93 3%) had

Their median age was 57 years, and 42 of 45 patients (93.3%) had a good performance status (ECOG 0 or 1). Ten patients had recurrent disease after previous curative gastrectomy and nine had previous adjuvant chemotherapy (three FAM, five doxifluridine or 5-FU plus cisplatin, and one doxifluridine plus mitomycin-C). The median disease-free interval of relapsed patients was 33.0 months (range=16.4�C55.6 months). Twenty-six patients (57.8%) had multiple metastases involving two or more organs, with the abdominal lymph nodes and liver being the most common sites of metastases. Table 1 Patient characteristics (n=45) Efficacy and survival A total of 43 patients were assessable for response (Table 2).

Of the two patients not assessable, one was lost to follow-up after the first cycle of treatment, and the other died after the first cycle of unknown cause, although brain metastasis was suspected. Of the 43 assessable patients, 2 achieved CR and 20 achieved PR, giving an overall response rate of 48.9% (95% CI=30.3�C63.5%) in the ITT population. Table 2 Antitumour activity There was no difference in overall response rate between patients who were pretreated and who were not with adjuvant chemotherapy (33.3 vs 52.8%, P=0.459 by Fisher’s exact test). The median duration of response in the 22 responding patients was 6.1 months (range=2.9�C12.4 months). The median follow-up period was 42.2 months (range=31.2�C54.3 months). The median TTP for all patients was 5.6 months (95% CI=3.9�C7.2 months) (Figure 1), and the median OS was 11.3 months (95% CI=8.1�C14.

4 months) (Figure 2), with a 1-year survival rate of 46.0% (95% CI=31.4�C60.6%). Figure 1 Time to progression for all patients. Figure 2 Overall survival for all patients. Poststudy treatment After disease progression, 28 patients (62.2%). received second-line chemotherapy, most commonly with irinotecan (21 patients; 75%) in combination with cisplatin/mitomycin-C or 5-FU/LV. Two patients (7.1%) achieved PR and 5 (17.9%) had stable disease in response to second-line chemotherapy, the median TTP was 1.5 months (95% CI=1.3�C1.8 months). Batimastat Three patients (6.7%) received palliative radiotherapy, and one patient underwent total gastrectomy due to cancer bleeding. Adverse events A total of 248 treatment cycles (median=6; range=1�C9 cycles) were administered, with 246 cycles and 43 patients assessable for safety. The frequencies of haematological and non-haematological adverse events are shown in Table 3 and and4,4, respectively. The most common haematological adverse event was neutropaenia, which occurred at grade 3 or 4 intensity in 20 patients (46.5%) and during 51 cycles (20.7%). No patient experienced febrile neutropaenia. There were no treatment-related deaths.

Research frontiers This is the first report about the role of Rec

Research frontiers This is the first report about the role of Recql5, a DNA helicase that plays an important role in the maintenance of genome integrity, Imatinib side effects in suppressing tumorigenesis in the gastrointestinal (GI) tract in mice. Innovations and breakthroughs In this article, the authors reported that Recql5 has a role in suppressing tumorigenesis in the GI tract in mice. Since mouse Recql5 and its human homologue (RECQL5) are highly conserved, these new findings have implicated RECQL5 as a suppressor for colorectal cancer in humans. Thus, RECQL5 may be used as a biomarker for this disease. Moreover, they have recently shown that mutations in Recql5 resulted in a significantly enhanced sensitivity to anticancer drug camptothecin, the prototype of irinotecan that is currently used to treat colorectal cancer patients.

Thus, the expression of RECQL5 could be used as a criterion for selecting patients for irinotecan-based chemotherapies. Applications No direct application may be derived based on the findings from this study. However, these findings should justify further investigation of the potential role of RECQL5 mutations in human GI cancers and the potential use of RECQL5 as a colorectal cancer biomarker or drug target. Peer review This is a well conceived, concisely written article, which certainly deserves publication. Acknowledgments We are grateful to Dr. Hua Lou for his comments on the manuscript and to Ellen L Barnes for her technical assistance in this work. Footnotes Supported by Grants RO1 CA88939, P20 CA103736 from the US National Institutes of Health and Searle Scholar Award 01-E-109 from the Searle Scholar Program Peer reviewers: Dr.

Inti Zlobec, PhD, Institute for Pathology, University Hospital Basel, Schoenbeinstrasse 40, Basel, CH-4031, Switzerland; Nathalie Wong, PhD, BSc (Hons), Professor, Department of Anatomical and Cellular Pathology, The Chinese University of Hong Kong, Shatin NT, Hong Kong, China; Ralph Graeser, PhD, Group Leader, Molecular & Cellular Biology, ProQinase GmbH, Breisacher Str. 117, Freiburg, 79106, Germany S- Editor Tian L L- Editor Logan S E- Editor Zheng XM
Now, more than ever, clinical oncologists are struggling to optimize treatment in cancer patients. With the use of molecular targeted agents and the incorporation of pharmacogenetics and pharmacogenomics in basic cancer treatment, a meaningful relationship between genotype (polymorphisms and mutations), gene expression profiles (level of gene expression of all or of target genes in the genome) and phenotype is being established, aimed at interpreting the variability among individuals in terms of response, Brefeldin_A resistance and toxicity to different drugs[1,2]. Pharmacogenetics (e.g.

Strikingly, both mTORC1 target genes and mTOR mRNA itself were in

Strikingly, both mTORC1 target genes and mTOR mRNA itself were induced in progressive diseases, whereas transcript levels were unchanged or even repressed in MCD compared with controls (Figure (Figure6A).6A). Consistent with this finding, immunofluorescence staining of biopsy samples for phosphorylated selleck kinase inhibitor S6 (pS6) as a marker of mTORC1 activity confirmed increased pS6 levels in podocytes of patients with diabetic nephropathy (Figure (Figure6,6, B and C). Profound activation of mTORC1 and phosphorylation of S6 was also observed by immunofluorescence staining and Western blot analysis of pS6 in an animal model of diabetic nephropathy (streptozotocin [STZ] model) (Figure (Figure6,6, D�CF), confirming the association of mTORC1 activation and diabetic nephropathy.

Figure 6 mTORC1 hyperactivation is a molecular signature of diabetic nephropathy. Podocyte-specific genetic inhibition of mTOR activation prevents progressive glomerular diseases. We next investigated whether interference with mTORC1 activation ameliorates the progression of renal disease involving the podocyte. Since complete deletion of mTORC1 in podocytes resulted in glomerular disease, we generated mice lacking only one Raptor allele in podocytes (RaptorHet podocyte) (Figure (Figure7A).7A). Western blot analysis from glomerular lysates confirmed a reduction of raptor protein and pS6 levels in RaptorHet podocyte mice (Figure (Figure7,7, B and C). To induce diabetic nephropathy, mice were injected with low-dose STZ at 8 weeks of age. Blood glucose levels, blood pressure, and glomerular macrophage infiltration were similar in RaptorHet podocyte and control mice (Supplemental Figure 4).

However, induction of diabetes resulted in a significant increase of mTORC1 activity, as evidenced by increased signals of pS6 in diabetic WT animals; pS6 levels were significantly lowered by deletion of 1 Raptor allele in RaptorHet podocyte mice (Figure (Figure7,7, D and E). While WT diabetic mice developed substantial proteinuria by 20 weeks of age, proteinuria was significantly reduced in diabetic RaptorHet podocyte mice (Figure (Figure7F).7F). Strikingly, diabetic RaptorHet podocyte mice displayed significantly reduced glomerulosclerosis scores and less mesangial matrix expansion (Figure (Figure7,7, G�CI).

Quantitative stereological analyses demonstrated that the mean podocyte volume in RaptorHet podocyte mice was reduced by about 30% compared with that in diabetic WT animals (Figure (Figure7,7, J Dacomitinib and K). It is worth noting that similar results were also observed in animal models of type 2 diabetes (36), underlining that mTORC1 deregulation is a major driving force for glomerular diseases such as diabetic nephropathy, which can be prevented by lowering mTOR activity in podocytes. Figure 7 Podocyte-specific genetic inhibition of mTOR hyperactivation prevents progressive glomerular diseases.

In addition to observations in pancreatic carcinomas, Smad4

In addition to observations in pancreatic carcinomas, Smad4 selleckchem is also known as a gene involved in juvenile polyposis tumour predisposition syndrome (Howe et al, 1998; Huang et al, 2000). Mutations of the Smad4 gene have been detected in some colorectal cancers, but its role in this specific cancer remains unclear. The frequencies of mutations (5�C45%) have been found to be low (Takagi et al, 1996; MacGrogan et al, 1997; Ohtaki et al, 2001), but data originated from relatively small studies, and the tumour populations examined were inhomogeneous explaining the broad range of incidences found. The aim of this study was to further expand these data by Smad4 mutation analysis of a large set of early-stage (I�CIII) colorectal cancer patients treated in a randomised multicentre trial of 5-fluorouracil (5-FU)/Mitomycin C adjuvant chemotherapy of the Swiss Group for Clinical Cancer Research (SAKK study 40/81).

Owing to the significance of LOH in colorectal cancer and the role of the remaining gene, this study was focused on patients with an allelic loss of one Smad4. METHODS Patients Patients from whom biopsies were isolated, were part of a previous randomised multicentre study of the SAKK on the benefit of treatment with adjuvant chemotherapy between 1981 and 1987 (Laffer, 1995). Deoxyribo nucleic acid (DNA) samples of these patients were extracted from tumour as well as from healthy tissue derived from the same patient in order to perform genetic analyses. Paraffin-embedded material was available from 329 of the 505 patients.

To investigate genetic alterations in the 18q21 region in these tumours, a gene dosage study of the tumour-suppressor genes Smad2, Smad4 and DCC was performed (Boulay et al, 1999). For technical reasons, high-quality DNA for analysis was available from 294 patients only. Individual dosage of the Smad4 gene showed a total deletion frequency (one or both alleles) of 68% when compared to normal tissue. In total, 167 patients (=57%) were detected with an allelic loss of one Smad4 copy. In this study, we randomly chose 73 of these 167 patients to search for the presence of point mutations in the remaining gene. After analysis of these 73 out of 167 patients, two point mutations of Smad4 had been detected, and for statistical reasons, further mutation analysis in the remaining 94 out of 167 patients did not seem necessary to substantiate our finding.

Gene copy status scoring Genomic samples from 294 patients were tested for copy dosage of the Smad4 gene using TaqMan quantitative real-time PCR (Perkin-Elmer, GSK-3 Huenenberg, Switzerland). Copy status of the Smad4 gene was determined by comparing tumour DNA to DNA from normal tissue derived from the same patient as described previously (Boulay et al, 1999). Duplex PCR Polymerase chain reaction (PCR) amplification on DNA was performed in 15��l reaction volume, containing 1.