Four subtypes have been detected, and pathogenicity, zoonotic potential, or stability may vary between subtypes. Non–travel-related hepatitis E virus (HEV) genotype 3 infections in persons in the Netherlands may have a zoonotic, foodborne, or water-borne origin. Possible reservoirs for HEV transmission by water, food, and animals were studied. HEV genotype 3/open reading frame 2 sequences were detected in 53% of pig farms, 4% of wild boar feces, and 17% of surface water samples. HEV sequences grouped within 4 genotype 3 clusters, of which 1 is so far unique to the Netherlands. The 2 largest clusters contained 35% and 43% of the animal and environmental sequences and 75% and 6%, respectively, of human HEV sequences obtained from a study on Dutch hepatitis E patients. This finding suggests that infection risk may be also dependent on transmission routes other than the ones currently studied. Besides the route of exposure, virus characteristics may be an important determinant for HEV disease in humans. Hepatitis E virus (HEV) is an RNA virus that causes liver inflammation in humans, predominantly in developing countries. In the 1990s, serologic studies among blood donors in industrialized countries showed that anti-HEV seropositivity also occurred among a small percentage (1.1%–1.4%) of persons without a travel history to a hepatitis E–endemic region  that were caught in 2 regions in the Netherlands, in the northeast (Groningen) and in the southeast (Limburg)  throughout 2005  were collected from the Meuse River monthly by using a conventional filter adsorption–elution method and concentrated by ultrafiltration by using a cellulose-acetate filter  under high pressure (3 bar)  was performed on 10-fold serially diluted RNA samples with primers HEVORF2con-s1 and HEVORF2con-a1, which were specific for the conserved open reading frame (ORF) 2 region, as described  and corresponded with analysis of a 242-nt fragment of ORF1 and a 148-nt fragment of ORF2. Human HEV sequences were obtained from human serum samples used for diagnosis of acute viral hepatitis (HEV RT-PCR products positive by hybridization (ORF2) or of the correct size (ORF1) were subjected to electrophoresis on a 2% agarose gel, excised, purified by using a Qiaquick Gel Extraction Kit , and sequenced by using the BigDye Terminator Cycle Sequencing Ready Reaction Kit . HEV RT-PCR products for which no sequences were obtained by direct sequencing were cloned into a pCRII-TOPO vector ; Ten-fold serially diluted RNA samples extracted from animal samples and river water were analyzed for HEV ORF2 sequences by RT-PCR. The highest prevalence of HEV ORF2 RNA in 51 (53%) of 97 pooled fecal samples was detected in pig farms housing pigs 5–27 weeks of age . A preva3 to 106 PDU/g. In 2 (17%) of 12 river water samples analyzed, HEV RNA was detected at concentrations of 2 PDU/L to 100 PDU/L.HEV RNA concentrations in positive fecal samples varied from 10www.ncbi.nlm.nih.gov) by phylogenetic analysis. This comparison showed that all belonged to HEV genotype 3.Thirty-six newly generated nucleotide sequences of 148 nt of HEV ORF2 were obtained from pooled fecal samples from pig farms that were previously shown to be positive for HEV ORF1 sequences . These sSequences grouped within 4 previously proposed genotype 3 clusters  , of whicTwo sequences in cluster 3f obtained from different pig farms were 100% identical within the 148-nt fragment of ORF2. A total of 242 nt of ORF1 were sequenced and showed that the 2 sequences were 98.8% identical within this region of the HEV genome. This result indicated that the strains were similar but not identical.Dutch environmental and animal HEV sequences obtained during 2004–2006 were compared with HEV ORF2 sequences from a study on Dutch hepatitis E patients without a travel history who received a diagnosis during 2004–2006  (19). NuHuman sequences grouped within the same 4 HEV genotype 3 clusters, with most of the sequences clustering within Dutch cluster 3c. A relatively high percentage of the sequences in cluster 3c is of human origin (43%). In this cluster, 1 human sequence was 100% identical with a porcine HEV sequence. For confirmation of homology, ORF1 RT-PCRs were performed, but only the pig sample yielded an ORF1 sequence. No direct geographic evidence could be established for an association between the patient and the pig farm; the patient who contracted the HEV infection in 2005 lived in the northern region of the Netherlands, but the pig farm was located in the eastern region. Comparison of postal codes of other Dutch patients without a recent travel history with postal codes of environmental sample locations did not show any geographic clustering.In cluster 3f, only 1 (5%) of 21 sequences identified during 2004–2006 was of human origin. Comparison with HEV sequences derived from human serum samples showed 2 close homologies, 1 between sequences from a hepatitis E patient in 2005 and a pig (96.6%) and 1 between sequences from a patient in 2006 and a surface water sample in 2005 (97.3%).Evidence was obtained for the presence of HEV in food, water, and animals in the Netherlands. Although 4 genotypes of HEV are known, 3 of which have been detected in pigs, each HEV ORF2 fragment sequenced in our study was identified as genotype 3. The highest prevalence (53%) was found on pig farms housing pigs 5–27 weeks of age, which is consistent with studies reporting that most HEV infections in pigs occurred between 2 and 4 months of age   virus. To confirm this hypothesis, samples should be tested for infectious virus by in vivo infection experiments  showed large differences (43% vs. 5%). This finding suggests that HEV 3c strains may be more pathogenic to humans, more stable in the environment, or are shed in higher numbers. Strains of feline calicivirus with different pathogenicities obtained from distinct outbreaks did not show conserved changes in virus genomes. Nevertheless, strains with higher pathogenicity infected tissue culture cells more efficiently and showed earlier cytopathic effects   suggest that the route of exposure and the virus subtype will play a role in HEV infection and disease in humans.


In a series of 46 glioblastomas, 16 anaplastic astrocytomas and eight astrocytomas, all tumours retaining one or both alleles of CDKN2A (48 tumours) and CDKN2B (49 tumours) were subjected to sequence analysis (entire coding region and splice acceptor and donor sites). One glioblastoma with hemizygous deletion of CDKN2A showed a missense mutation in exon 2 (codon 83) that would result in the substitution of tyrosine for histidine in the protein. None of the tumours retaining alleles of CDKN2B showed mutations of this gene. Glioblastomas with retention of both alleles of CDKN2A (14 tumours) and CDKN2B (16 tumours) expressed transcripts for these genes. In contrast, 7/13 glioblastomas with hemizygous deletions of CDKN2A and 8/11 glioblastomas with hemizygous deletions of CDKN2B showed no or weak expression. Anaplastic astrocytomas and astrocytomas showed a considerable variation in the expression of both genes, regardless of whether they retained one or two copies of the genes. The methylation status of the 5' CpG island of the CDKN2A gene was studied in all 15 tumours retaining only one allele of CDKN2A as well as in the six tumours showing no significant expression of transcript despite their retaining both CDKN2A alleles. Three tumours  were found to have partially methylated the 5' CpG island of CDKN2A. It appears that in human astrocytic gliomas point mutations of the CDKN2A and CDKN2B genes are uncommon and hypermethylation of the 5' CpG region of CDKN2A does not appear to be a major mechanism for inhibiting transcription of this gene.


LKB1 and SMAD4 genes have been shown recently to cause a number of PJS and JPS cases respectively, but there remains considerable uncharacterized genetic heterogeneity in these syndromes, particularly JPS. The mouse homologue of CDX2 has been shown to give rise to a phenotype which includes hamartomatous-like polyps in the colon and is therefore a good candidate for JPS and PJS cases which are not accounted for by the SMAD4 and LKB1 genes. By analogy with SMAD4CDX2 is also a candidate for somatic mutation in sporadic colorectal cancer. We have screened 37 JPS families/cases without known SMAD4 mutations, 10 Peutz-Jeghers cases without known LKB1 mutations and 49 sporadic colorectal cancers for mutations in CDX2. Although polymorphic variants and rare variants of unlikely significance were detected, no pathogenic CDX2 mutations were found in any case of JPS or PJS, or in any of the sporadic cancers. © 2001 Cancer Research Campaign www.bjcancer.comPeutz–Jeghers syndrome (PJS) and juvenile polyposis (JPS) are both characterized by the presence of hamartomatous polyps and increased risk of malignancy in the gastrointestinal tract. Mutations of the


We followed 87 cirrhotic patients with esophageal varices and without previous hemorrhage for a mean period of 24 mo to prospectively evaluate the occurance of variceal bleeding within (early) or after (late) 6 mo from entry and the contribution of portal Doppler ultrasound parameters to the prediction of early and late hemorrhage. Clinical, biochemical, endoscopic and portal Doppler ultrasound parameters were recorded at entry. Variceal bleeding occurred in 22 patients (25.3%). Nine (40.9%) bled within the first 6 mo. Cox regression analysis identified variceal size, cherry-red spots, serum bilirubin and congestion index of the portal vein  as the only independent predictors of first variceal hemorrhage. Discriminant analysis was used to find the prognostic index cut off points to identify patients who bled within 6 mo (prognostic group 1) or after 6 mo (prognostic group 2) or remained free of bleeding (prognostic group 3). The cumulative proportion of patients correctly classified was 73% in prognostic group 1, 47% in prognostic group 2 and more than 80% in prognostic group 3. The addition of Doppler ultrasound flowmetry to clinical, biochemical and endoscopic parameter only improved the classification of patients with early bleeding.


The aim of the study was to determine the relationship between StO2 and mixed venous oxygen saturation (SvO2) in patients with severe left heart failure with or without additional severe sepsis or septic shock.Low cardiac output states such as left heart failure are characterized by preserved oxygen extraction ratio, which is in contrast to severe sepsis. Near infrared spectroscopy (NIRS) allows noninvasive estimation of skeletal muscle tissue oxygenation (StOn = 24) and B (n = 41) included patients without and with additional severe sepsis/septic shock, respectively. Thenar muscle StO2 was measured using NIRS in the patients and in 15 healthy volunteers.Sixty-five patients with severe left heart failure due to primary heart disease were divided into two groups: groups A . StO2 was higher in group B than in healthy volunteers (P = 0.02). In group A StO2 correlated with SvO2 , although StO2 overestimated SvO2 . In group A changes in StO2 correlated with changes in SvO2 . In group B important differences between these variables were observed. Plasma lactate concentrations correlated negatively with StO2 values only in group A .StO2 does not estimate SvO2 in patients with severe left heart failure and additional severe sepsis or septic shock. However, in patients with severe left heart failure without additional severe sepsis or septic shock, StO2 values could be used to provide rapid, noninvasive estimation of SvO2; furthermore, the trend in StO2 may be considered a surrogate for the trend in SvO2. Skeletal muscle StOTrial Registration: NCT00384644 In 80% of patients pathogenic bacteria were isolated.Included in the study were 65 patients  with primary heart disease . In 24 patients (group A) severe left heart failure or cardiogenic shock but no additional severe sepsis/septic shock was the reason for ICU admission. In 25 patients severe sepsis and in 16 patients septic shock was diagnosed . StO2 was higher in group B patients than in healthy volunteers (P = 0.02). In group A StO2 correlated with SvO2 , but no correlation was observed between StO2 and SvO2 in group B  was detected.In group A StO2-StO2 changes were recorded. Changes in StO2 correlated with changes in SvO2 ; the equation for the regression line was as follows  = 0.84 × ΔStO2 (%) - 0.67. In eight patients from group B 38 pairs of SvO2-StO2 changes were recorded. In group B changes in StO2 did not correlate with changes in SvO2 .In 10 patients from group A 42 pairs of SvO2 values in group A ; there was no correlation between lactate and StO2 in group B.Plasma lactate concentrations correlated negatively with StO2 does not estimate SvO2 in patients with severe left heart failure and additional severe sepsis or septic shock. However, in patients with severe left heart failure without additional severe sepsis or septic shock, the StO2 value could be used as a fast and noninvasive estimate of SvO2; also, the trend in StO2 may be considered a surrogate for the trend in SvO2.The main result of the study is that skeletal muscle StO2 and slow deceleration in StO2 during stagnant ischaemia in septic patients [per se, because an increase in tissue oxygen tension is normally observed [2 levels seen in our patients with additional severe sepsis or septic shock support this hypothesis. Mitochondrial dysfunction has been implicated by Ince and Sinaasappel [We previously detected high StOpatients . Our datpatients . Studiespatients . Howeverpatients . Reducedobserved . The higaasappel . This miaasappel .2/low SvO2, seen in severe sepsis and septic shock, suggest blood flow redistribution. Thenar muscle StO2 probably correlates with central venous oxygen saturation (ScvO2), which is measured in a mixture of blood from head and both arms. In healthy resting individuals ScvO2 is slightly lower than SvO2 [2 is greater than ScvO2.The high StOhan SvO2 . Blood ihan SvO2 . As a re2 and SvO2 in both haemodynamically stable and shocked patients. In stable patients ScvO2 was similar to SvO2. In patients with failing heart ScvO2 was slightly higher than SvO2 and in shock patients the difference between SvO2 and ScvO2 was even more pronounced . Lee and coworkers [2 measurements could substitute for SvO2 demonstrated problematic large confidence limits [This relationship changes in the presence of cardiovascular instability. Scheinman and coworkers  performeoworkers  describee limits  and poore limits .2 is greater than SvO2 [2 (or probably StO2) as a treatment goal, the relative oxygen consumption of the superior vena cava system may remain stable at a time when oxidative metabolism of vital organs, such as the splanchnic region, may reach a level at which flow-limited oxygen consumption occurs, together with marked decrease in oxygen saturation. In this situation StO2 provides a falsely favourable impression of adequate body perfusion, because of the inability to detect organ ischemia in the lower part of the body.Most authors attribute this pattern to changes in the distribution of cardiac output that occur in the presence of haemodynamic instability. In shock states, blood flow to the splanchnic and renal circulations falls, whereas flow to the heart and brain is maintained . This rehan SvO2 -25. Cons2 of 75% or less ; they were all in septic shock  with low cardiac index (< 2.0 l/min per m2). These patients were probably in an early under-resuscitated phase of septic shock. Low numbers of septic patients with low StO2 values did not allow us to study the agreement between StO2 and SvO2 in such patients; however, there was a wide range in StO2 values with SvO2 below 65%. Additional research is necessary to study muscle skeletal StO2 in under-resuscitated septic patients.In the present study three patients with septic shock had skeletal muscle StO2  and systemic vascular resistance . No correlation was found between systemic oxygen transport variables and skeletal muscle partial oxygen pressure in septic patients. These measurements were taken in the most common cardiovascular state in sepsis; this is in contrast to hypodynamic shock, which is only present in the very final stages of sepsis or in patients without adequate volume replacement [Our data are supported by previous work conducted by Boekstegers and coworkers , who mealacement . In a sulacement .2 and venous oxygen saturation  was observed; the venous effluent was obtained from a deep forearm vein that drained the exercising muscle. StO2 was minimally affected by skin blood flow. Changes in limb perfusion affect StO2; skeletal muscle StO2 decreases during norepinephrine and increases during nitroprusside infusion.In a human validation study  a signif2  correlated with systemic DO2 in a pig model [2 is already used in trauma patients, in whom it identifies the severity of shock [2 can track systemic DO2 during and after resuscitation of trauma patients [In shock with preserved or even increased oxygen extraction, such as haemorrhagic shock, StOig model . Changesig model . Continuof shock . Basal spatients .2 overestimated SvO2 (bias -2.5%) in the present study. This may be due to the NIRS method, which does not discriminate between compartments. It provides a global assessment of oxygenation in all vascular compartments  in sample volume of underlying tissue. This is major limitation of the present study. The noninvasive measurement of only venous oxygen saturation is complicated by the fact that isolation of the contribution of venous compartment to the noninvasive optical signal is not straightforward. New methods like near-infrared spiroximetry, which measures venous oxygen saturation in tissue from the near-infrared spectrum of the amplitude of respiration-induced absorption oscillations, may lead to the design of a noninvasive optical instrument that can provide simultaneous and real-time measurements of local arterial, tissue and venous oxygen saturation [StOturation .2 regional monitoring to guide tissue oxygenation, in addition to the early goal-directed therapy proposed by Rivers and coworkers [In low flow states, in which controversies regarding monitoring persist , it appeoworkers .2 provides a noninvasive estimate of and tracks with StO2. It should be emphasized that in patients with severe heart failure and additional severe sepsis or septic shock, skeletal muscle StO2 provides a falsely favourable impression of body perfusion.In patients with severe left heart failure without additional severe sepsis or septic shock, SvO2 does not estimate SvO2 in patients with severe left heart failure and additional severe sepsis or septic shock.• Skeletal muscle StO2 values could be used to provide rapid, noninvasive estimation of SvO2; furthermore, the trend in StO2 may be considered a surrogate for the trend in SvO2.• StO2 = oxygen distribution; ICU = intensive care unit; NIRS = near infrared spectroscopy; ScvO2 = central venous oxygen saturation; SOFA = Sepsis-related Organ Failure Assessment; StO2 = tissue oxygenation; SvO2 = mixed venous oxygen saturation.DOThe authors declare that they have no competing interest.MP was responsible for conception and design of the study; for acquisition of data, and its analysis and interpretation; and for drafting the manuscript. HM was responsible for conception and design of the study; for acquisition of data, and its analysis and interpretation; and for drafting the manuscript.


The mapping of allelic loss on the short arm of chromosome 1 has been performed in non-small-cell lung cancer. We used a set of 11 microsatellite loci spanning 1p to examine the frequency of allelic imbalance in a panel of 58 tumours. Fifty-one of 58 (87.9%) cases have shown somatic allelic loss at one or more loci tested. The two shortest regions of the overlap (SRO) of the deletions have been identified: SRO 1 at 1p13.1 and SRO 2 at 1p32-pter. Allelic losses at these regions have been compared among adenocarcinoma and squamous cell carcinoma and no difference has been found. In contrast to SRO 1, deletions at SRO 2 significantly correlated with advanced stage of the disease as well as post-operative metastasizing and relapse. These data may suggest that SRO 1 and SRO 2 can harbour tumour-supressor genes (TSGs) involved in different stages of NSCLC development. SRO 2 is still quite large and its refined mapping should help attempts to clone and identify the putative TSG(s). Microsatellite instability (replication errors) affecting only 6 (10.3%) of 58 tumour samples is an infrequent genetic alteration at the loci tested.


In order to assess the role of endoscopic retrograde cholangiography in evaluating the patients with post-operative biliary leak and of endoscopic nasobiliary drainage in its management, 36 patients with biliary leak seen over a period of 9 years were studied. Thirty-two had biliary leak following cholecystectomy, 3 following repair of liver trauma and 1 following choledochoduodenostomy. Patients presented at an interval of 4 days to 210 days  following laparotomy. Hyperbilirubinemia was noticed in only 13 patients (36.1%), while abdominal ultrasonogram showed ascites or biloma in 24 (66.7%). Endoscopic retrograde cholangiography showed the leak to involve the common bile duct in 55.6%, cystic duct in 33.3% and intrahepatic biliary radicles in 8.3%. Associated lesions included bile duct obstruction due to stricture or accidental ligature in 20%, bile duct stone in 20% and liver abscess in 2.8%.        Endoscopic nasobiliary drainage using a 7 Fr pig-tail catheter was attempted in 14 patients and could be established in 12 of them. Bile duct leak sealed in all but one of these 12 patients after an interval of 3 days to 40 days . A single patient with large defect and a proximal bile duct stricture did not respond and required surgery. Common bile duct stones were removed by endoscopic sphincterotomy in 3 out of 4 patients. One patient with large stone required surgical choledocholithotomy. In conclusion, endoscopic retrograde cholangiography was safe and useful in confirming the presence of leak as well as its site, size and associated abnormalities. Endoscopic nasobiliary drainage proved an effective therapy in post-operative biliary leak and could avoid re-exploration in 71.4% patients.


The results implied possible role of IRF2 in controlling the intricate balance of cellular gene expression under stress conditions in general. Here we have investigated the secondary structure of the Internal Ribosome Entry Site of IRF2 RNA and demonstrated the role of PTB protein in ribosome assembly to facilitate internal initiation.Earlier we have reported translational control of interferon regulatory factor 2 (IRF2) by internal initiation . Deletion of 25 nts from the 3′terminus of the 5′untranslated region resulted in reduced binding with PTB protein and also showed significant decrease in IRES activity compared to the wild type. We have also demonstrated putative contact points of PTB on the IRF2–5′UTR using primer extension inhibition assay. Majority of the PTB toe-prints were found to be restricted to the 3′end of the IRES. Additionally, Circular Dichroism (CD) spectra analysis suggested change in the conformation of the RNA upon PTB binding. Further, binding studies using S10 extract from HeLa cells, partially silenced for PTB gene expression, resulted in reduced binding by other trans-acting factors. Finally, we have demonstrated that addition of recombinant PTB enhances ribosome assembly on IRF2 IRES suggesting possible role of PTB in mediating internal initiation of translation of IRF2 RNA.We have probed the putative secondary structure of the IRF2 5′UTR RNA using various enzymatic and chemical modification agents to constrain the secondary structure predicted from RNA folding algorithm Mfold. The IRES activity was found to be influenced by the interaction of trans-acting factors to mediate internal initiation of translation.It appears that PTB binding to multiple sites within IRF2 5′UTR leads to a conformational change in the RNA that facilitate binding of other  IRF1 function as an activator of IFN and IFN-inducible genes, whereas IRF2 act as repressor of IRF1 action trans-acting factors. Ribosome binding analysis also indicated that supplementation of purified PTB enhanced the formation of translation initiation complex. This led us to hypothesize that PTB interaction with the IRF2 IRES might trigger a change in the conformation that helps in the recruitment of other hitherto unknown trans acting factors and ribosomes, thus facilitating translation initiation.We have partially mapped the secondary structure of the IRF2 5′UTR using various enzymatic and chemical modification probes. The data validates the predicted MFOLD structure of the 5′UTR RNA. To map the putative contact points of the protein on the IRF2 5′ UTR we have performed primer extension inhibition assay. PTB was found to have multiple contact points on the IRF2 RNA, but mostly at the 3′end of the 5′UTR. Since many contact points were found on the 3′ end of the RNA, a deletion mutant was generated, where 25 nt from 3′end was deleted. UV cross-linking and competition experiments showed a reduced binding of the purified PTB on the mutant RNA as compared to the wild type RNA. The above data was supported by Circular Dichroism (CD) spectra analysis of the RNA, which also suggested a change in the conformation of IRF2 5′ UTR in presence of PTB binding. Proteins from whole cell extracts showed that full length IRF2 IRES interacted predominantly with a 57 kDa (PTB) protein, compared to the mutant IRF2. Further PTB silencing experiments showed reduced PTB binding along with significant reduction in binding of other cellular Earlier we have shown that the putative secondary structure of IRF2 5′UTR RNA (predicted by using the MFOLD program) IRF2 monocistronic RNA was generated from the corresponding DNA construct . The RNAin vitro transcribed IRF2 UTR RNA was incubated with RNase T1 followed by extension using AMV-RT (Promega). The products were analyzed on 8% polyacrylamide-8M urea denaturing gel. For precise mapping of the contact points, a DNA sequencing reaction with the same end labeled primer was run alongside . Since IRF2 5′UTR folds into a fairly stable secondary structure, only few specific RT pauses were observed at G8, G44, G59, G69, G83 and G131 , the pauses corresponding to nuclease (TI and VI) cleavage sites and the toe prints corresponding to PTB binding sites were mapped to MFOLD predicted structure for clarity . The res32P labeled wild type IRF2 5′UTR RNA was incubated with 0.87 nM of purified PTB protein in absence and presence of increasing concentrations of the competitor RNAs. The toe-printing data showed that the contact points of PTB are predominantly at the 3′ end of the IRF2 5′UTR RNA. To investigate this further, 25 nucleotides were deleted at the 3′end from the IRF2 5′ UTR to generate mIRF2 . To compSince 3′ deletion mutant showed reduced PTB binding, we hypothesized that this might also affect the IRF2-IRES activity. To investigate it, the deletion mutant was cloned in the bicistronic plasmid construct . Both wiTo further verify the possible conformational change in the RNA upon protein binding CD spectroscopic studies were performed. CD spectra of IRF2 5′UTR RNA was analyzed in absence and presence of purified recombinant PTB protein. CD spectra were obtained in 240 to 320 nm range at 20°C in 0.5 ml RNA binding buffer. The results showed distinct change in the wavelengths at which the peak of ellipticity value is reached for RNA incubated with PTB (260 nm) compared to RNA alone (256 nm). Also, at this particular wavelength there was significant increase in the ellipticity value suggesting a change in the conformation of the IRF2 5′UTR RNA in presence of PTB . Interes32P labeled Wt and mIRF2 UTR RNAs were incubated with the extracted total proteins, UV-crosslinked and analysed on SDS-PAGE. The results indicated that wtIRF2 5′UTR interacted strongly with 57 kDa protein (PTB), whereas with mIRF2 5′UTR the intensity of the band corresponding to 57 kDa protein was relatively less. Interestingly, the mIRF2 showed binding with additional proteins, some of those could be the cleavage products of PTB (p25), which might negatively influence IRES activity trans acting factors to enhance translation, PTB depleted HeLa S10 was used in UV cross linking study. PTB was partially silenced in HeLa cells by using increasing concentration of siPTB. 36 hours post transfection S10 extracts were prepared. A significant decrease of PTB protein level was observed at 60 nm & 80 nm concentration of siPTB. UV-crosslinking experiment was performed using wt IRF2 RNA probes and S10 extracts from control or siPTB treated cells. Interactions of several cellular proteins were found to be significantly reduced  has been implicated as an important ITAF for a number of viral as well as cellular IRESs. It is believed that PTB has a chaperone like activity that can bring about a change in the conformation of the IRES structure, which might facilitate the interaction of the ribosomes with the RNA leading to efficiently initiate translation.The mechanism of IRES mediated translation in cellular RNA is still not completely understood. Various IRES In this paper, we have investigated the interaction of PTB with the IRF2-IRES RNA and studied its importance on its translation initiation. Multiple contact points were mapped on the IRF2 IRES RNA. A significant number of those contact points were mapped to the 3′ end of the IRES element. In fact, Apaf-1 IRES is also known to have two potential PTB binding sites at the 3′ end region However recent report suggests that the IRES activity might not completely depend on the overall structure but on different sequence modules that play important role in the ITAF recruitment trans acting factors near the vicinity of the translation start site that would facilitate the recruitment of the ribosomes to the RNA and thus facilitating translation initiation. To further consolidate the observation, S10 extract isolated from HeLa cells, partially silenced for PTB expression was used for binding using Wt IRF2 probe. Several cellular tran-acting factors were unable to bind Wt IRF2 in reduced presence of PTB. Interestingly, we found that supplementing the in vitro translation mix with purified PTB marginally enhanced the formation of the 48S ribosomal initiation complex.Nuclease mapping and chemical modifications partly revealed the secondary structure of the IRF2 5′UTR, which largely supports the MFOLD prediction. DMS modification interference assay indicated perturbation of the RNA structure in presence of PTB. The presence of PTB masked the RNA, rendering it inaccessible to DMS modifications at certain places. Also, a few new DMS modification sites were found under such condition, suggesting changes in local structure due to protein binding. CD spectroscopic analysis reinforces the idea of change in RNA conformation upon protein binding. In fact when CD experiment was performed with the mutant IRF2 IRES RNA and the PTB protein, change in molar ellipticity was found to be less than that obtained with the wild type RNA. PTB has been shown to have chaperone like activity, thus it would be interesting to study how its interaction with the IRF2 5′ UTR RNA and consequent change in the RNA conformation helps in ribosome assembly during internal initiation. Also it is possible that such a change in the conformation of the RNA might recruit certain other trans-acting factors that could facilitate IRES mode of translation initiation in the IRF2 mRNA. However, at this stage it is not clear how PTB or other IRES trans-acting factors (ITAFs) interaction helps in switching on the IRES activity of the cellular IRES elements. It could be a complex interplay of PTB and several other ITAFs. Future experiments would be directed to understand how the ITAFs binding directs the ribosome to land preferentially to the IRES site and translate by internal initiation under stress conditions.Thus we hypothesize that PTB serves as an ITAF which recruits other possible No, an ethics statement is not required for this work.The bicistronic constructs containing respective 5′UTR sequences of full length IRF2 (NCBI accession number NM_002199) and 3′ deletion IRF2  were cloned downstream of the landscape of structure derived form the inactive part of ΔEMCV IRES sequence between Renilla luciferase (RLuc) and Firefly luciferase (FLuc) genes, in HindIII and EcoRI sites. The construct pRΔEnullF HeLa S3 cells were maintained in DMEM (Invitrogen) with 10% fetal bovine serum . Cells were transfected with various bicistronic plasmids and pSV40β-gal using Tfx 20 reagent (Promega) and luciferase assay was performed using Dual luciferase assay reagent (Promega). Transfection of siRNA was performed in HeLa S3 cells growing in monolayer using Lipofectamine-2000 transfection reagent and optiMEM-I prepared without addition of antibiotic (Invitrogen). Cells were seeded onto 100 mm dishes in duplicates one day prior to transfection in similar manner. Transfections were performed with 40 nm, 60 nm and 80 nm of siPTB (Dharmacon) or 80 nm of nonspecific si (Dharmacon) diluted with optiMEM-I to a final volume of 100 µl followed by incubation at room temperature for 20 minutes. Subsequently, 4.9 ml of optiMEM-I was added to the transfection mixture, which was then layered onto cells. 4 hours later the medium was replaced with 9 ml of DMEM (with antibiotic) and 10% FBS. 36 hours post transfection the cells were washed with PBS, and were trypsinized followed by HeLa S10 extract preparation. S10 extract was prepared from HeLa cells as described before (22).32P labeled RNA probes corresponding to the 5′UTRs of IRF2 and deletion mutant IRF2 were made from their respective plasmid DNAs after linearizing with EcoRI and transcribed with T7 RNA polymerase and 10 µCi/µl of alpha 32P UTP (NEN) as per manufacturer's guidelines.The IRF2 5′UTR and mIRF2 RNA probes were made from IRF2Fluc and mIRF2Luc DNA plasmids respectively linearized with Eco RI and was transcribed by T7 RNA polymerase. Similarly, the E.coli (BL21 DE3) cells transformed with the expression vector. His-tagged protein was purified using Ni2+-nitrilotriacetic acid-agarose (Qiagen) under non-denaturing conditions and eluted with 250 mM imidazole.The expression of recombinant PTB (NCBI accession number NM_031990) from PET28a-PTB (a generous gift from Dr. J.G. Patton) was induced by 0.6 mM IPTG in in vitro transcribed IRF2 bicistronic RNAs were denatured at 65°C for 10 minutes and then cooled slowly to room temperature. The RNAs were then digested with 0.5 unit of RNase T1 (SIGMA) for 15 minutes at 37°C. After incubation the reaction mixture was treated with proteinase K followed by phenol:chloroform extraction and alcohol precipitation. For RNase V1 (Ambion) digestion, the RNA was incubated with 0.01 units of RNase V1 for 10 minutes at 30°C. After the incubation the reaction mixture was extracted with phenol: chloroform and alcohol precipitated. T1 digestions were carried out in the buffer containing 10 mM Tris.Cl (pH 7.5), 10 mM MgCl2, and 100 mM KCl. The DMS modification was carried out in the buffer containing 50 mM sodium cacodylate (pH 7.5), 5 mM MgCl2, 100 mM KCl at 30°C for 10 minutes using 1 µl of DMS diluted 1∶8 in absolute ethanol. The modified RNAs were precipitated with ethanol. The digested and the modified RNAs were reverse transcribed using AMV RT (Promega) with an end labeled primer annealing to 1–20 at the 5′ end of the firefly luciferase gene. The cDNAs were precipitated with 0.3 M sodium acetate and absolute ethanol and then resolved in a 8M urea-8% Acrylamide gel electrophoresis along with a reference sequencing reaction  using the same end-labeled primer. Similarly, purified PTB bound RNA in buffer was modified with 1 µl of DMS diluted 1∶8 in absolute ethanol at 30°C for 10 minutes The modified RNAs were precipitated with ethanol the modified RNAs were reverse transcribed using AMV RT (Promega) with an end labeled primer annealing to 1–20 at the 5′ end of the firefly luciferase gene and was analyzed as mentioned before.10 µg of in vitro transcribed RNA corresponding to the IRF2 bicistronic RNA (derived from the construct pRIRF2F) and binding reaction was performed in a final volume of 20 µl at 30°C for 20 minutes. Reaction where PTB was not added serve as a negative control. To the reactions, [32P]-end labeled primer complimentary to 20 nucleotides of the 5′ end of the Firefly luciferase was added and allowed to extend using 3 units of AMV-Reverse transcriptase (Promega) at 30°C for one hour. The cDNAs were alcohol precipitated, resuspended and compared with the dideoxynucleotide sequence ladders (obtained using same end-labeled primer) by electrophoresis on a 6% polyacrylamide/8M urea denaturing gel.Increasing concentration of purified his-tagged PTB protein (500 and 800 ng) was incubated with 5 pmoles of 2, 3.8% glycerol, 2 mM DTT, 0.1 mM EDTA). CD spectra were obtained in 240 to 320 nm range at 20°C with IRF2 5′UTR (400 ng) and 800 ng of PTB or bovine serum albumin (BSA).Measurements of CD spectra were performed with a Jasco J-715 spectropolarimeter. Spectra were obtained in 0.5 ml RNA-binding buffer . Briefly, [α-Protein concentration of the S10 extract prepared from HeLa cells were assayed by Bradford (Bio-Rad). Equal amounts of cell extracts were loaded on SDS-10% polyacrylamide gel. The resolved proteins were electro-transferred to nitrocellulose membranes. Samples were then analysed by mouse monoclonal anti-PTB antibody , followed by secondary antibody . Rabbit polyclonal anti-actin (Sigma) was used as control for equal loading of total cell extracts. Antibody complexes were detected using the enhanced chemiluminescence (ECL) detection kit (Amersham Antibodies).32P UTP and the 25 µl reactions containing 17.5 µl RRL (Promega), 1.5 µl of amino acid mix (Promega) and PTB was supplemented as indicated in the figure legends. Labeled RNA 100,000 cpm was used per reaction. The reactions were incubated at 30°C for 20 min. and the volume was made upto 150 µl with the sucrose density gradient buffer . The reactions were overlaid on 5–30% linear sucrose density gradients and centrifuged for 3 hrs at 30,000 rpm using SW41 rotor in Beckman ultracentrifuge. 200 µl fractions were collected from top of the radioactive tube. Counts of each fraction were measured and %cpm was plotted against the fraction number.IRF2 UTR RNA was labeled with We gratefully acknowledge members of our laboratory for their help and suggestions.


Human endocrine tumors often express the somatostatin receptors SSTR 1–5 with different intensity. It has been widely investigated their distribution in pituitary adenomas, brain tumors, adrenal tumors and neuroendocrine tumors in gastrointestinal tract (NET). Some of studies also concern the expression of SSTRs in thyroid tumors but they are mainly limited to parafollicular C cells – derived medullary thyroid carcinomas (MTC). Results of SSTR 1–5 detection in other thyroid pathologies like follicular adenomas and papillary cancers are still scarce and often controversial, depending of investigation method used. The aim of this study was to report the presence of all the 5 subtypes of SSTR (including 2A and 2B SSTR isoforms) in some surgically treated human thyroid tumors by means of immunohistochemistry and real-time PCR method and to correlate the results obtained with both techniques. SSTR 1 protein was expressed in 88.8% of investigated cases, SSTR 2A and 2B both in 44.4%, SSTR 3 in 55.5%, SSTR 4 in 11.2% and SSTR 5 in 33.3%. SSTR 1 is the dominant form in the thyroid gland tumor and hyperplasia. We found positive confirmation of both methods in 88.8% for SSTR 1, 2A, 3 subtypes, in 22.2% for SSTR 4 and in 100% for SSTR 5. It suggests that somatostatin multiligand analogs or selective SSTR 1 agonists may be used in thyroid tumors treatment. Somatostatin (SST) as well as its synthetic analogs are engaged in regulation of hormones secretion in different ways and this action depends on specific receptors expression which are present on the target cells. Five subtypes of the SST receptor have been identified so far, i.e. SSTR 1–5. SSTR 2 is present in two splicing variants (2A and 2B)  and are ex vivo in vitro method in detecting cellular distribution of SST receptors .SSTR 1 which was distributed both in the cytoplasm and the membranes or only in the cytoplasm was shown in 8  specimens  and in the remaining 4 cases  distribution of this SSTR subtype was fluctuated from weak to pale. In 4 specimens  strong to moderate staining of SSTR 2B was observed with both membranous distribution and in the area of the cell cytoplasm. In the other 4 cases the staining intensity was weak or pale. Staining for SSTR 3 was characterized by cytoplasmic distribution with various intensity. In 5  out of 9 examined specimens SSTR 3 was found with strong or moderate intensity in the cell cytoplasm and the remaining 3 cases  showed weak to pale intensity of staining  of investigated cases with exception of 1  specimen showing cytoplasmic distribution of this receptor subtype fluctuating from pale to strong (patient nr 2) . Another 5  cases demonstrated pale to moderate staining. In 1  specimen the intensity of staining was from pale to strong (patient nr 2).Immunoreactivity of investigated thyroid tissues was estimate in pathologically altered cells of thyroid cancers and benign lesions of the thyroid gland. The area of normal surrounding tissue was immunonegative in all investigated cases. Multiple SSTR subtypes were found to coexist in each case of investigated tissue Table . Most ofens Fig. . SSTR 2AWe consolidated result for endogenous control gene of hypoxanthine-guanine phosphoribosyltransferase (HPRT) as negative  and according to this all remaining mRNAs were calculated relative to the amount of HPRT and given in arbitrary units. The diagram presented below show the mean results of 3 experiments for each primer Fig. . SSTR 1 We demonstrated in our investigation that somatostatin subtype receptors are widely distributed in human thyroid diseases with different intensity and cellular localization. It is consists with earlier reports of England et al. who findWe proved that somatostatin receptor subtypes are frequently expressed in pathologically altered thyrocytes, in contrast to normal thyroid follicular epithelium. SSTR 1 protein was expressed in 77,7% of investigated cases, SSTR 2A and 2B both in 44,4%, SSTR 3 in 55,5%, SSTR 4 in 11,2% and SSTR 5 in 33,3%. SSTR 1 is the dominant form in the thyroid gland tumor and hyperplasia. A good correlation with SSTR 1 receptor gene expression was observed in 77,7% of investigated cases. Immunohistochemical estimation of SSTR 2A and 3 was agree with RT-PCR method in 77,7%, only 22,2% of results of SSTR 4 mRNA estimation were correlated with immunohistochemical staining and SSTR 5 mRNA was good correlated with IHC staining in 100% tissues. It suggests that somatostatin multiligand analogs or selective SSTR 1 agonists may represent a further useful approach for the thyroid tumors treatment. The expression of somatostatin receptor subtypes in thyroid tumors needs father studies concerning greater and more differentiated group of patients.mem: membranous localization; cytopl: cytoplasmatic localization; strong staining (+++), moderate staining (++), weak staining (+) and pale staining (+/-); SST: somatostatin; SSTR: somatostatin receptor subtype; RT-PCR: real time polimerase chain reaction; RT-PCR: reverse – transcriptase polimerase chain reaction; ISH: in situ hybridisation; IHC: immunohistochemistry; NET: neuroendocrine tumors in gastrointestinal tract; MTC: medullary thyroid carcinoma; MNG: multinodular goiter; mRNA: messenger ribonucleic acid; HPRT: hypoxanthine-guanine phosphoribosyltransferase control gene.The authors declare that they have no competing interests.HP carried out the immunohistochemistry studies and drafted the manuscript. TS obtained thyroid tissues during surgery. RK established histopathological diagnosis. EB carried out the molecular biology studies. MP supervised the experiences, helped to draft and revised the manuscript and provided general support. All authors read and approved the final manuscript.


Rats were prepared surgically so that peripheral intestinal lymph could be collected from them while a syngeneic tumour (the HSN sarcoma) was growing in each major Peyer's patch of the small intestine. Dendritic lymph cells were isolated from the lymph and injected i.p. into naive, syngeneic rats. Each of the 16 recipients received just under 10(6) such cells and was challenged 10 days later with a subcutaneous dose of 10(4) viable HSN cells. Six weeks after this challenge only 7 of the recipients had a tumour and these were small (mean weight 1.8 g), while 17 controls (which had each been treated with 10(6) thoracic duct lymphocytes from the same donors, and given the same challenge) all had large tumours (mean weight 8.8 g). The remaining 9 test rats were still free of tumours when they were killed and autopsied 4 months after challenge. Dendritic lymph cells from normal rats were 'sensitised' by incubating them overnight on a monolayer of HSN cells. They were then transferred to 5 naive recipients which received the usual challenge. Six weeks later they all had tumours (mean weight 1.3 g) but these were much smaller than those in the 5 controls (mean weight 9.3 g).


Based on the important role of CD44 splice variants in colorectal cancer progression and metastasis, we evaluated the use of CD44v6 expression to detect and assess the metastatic potential of colorectal tumour cells circulating in peripheral blood. A nested amplification was designed that allowed to detect 10–100 colon cancer cells. This assay was applied to blood samples from healthy donors. Strong signals were detected in all cases, indicating that it cannot be used to detect colorectal carcinoma cells in whole blood. We then included an enrichment step based on the use of an anti-epithelial cells monoclonal antibody (BerEP4) coupled to magnetic beads. The CD44v6 reverse transcription polymerase chain reaction (RT PCR) assay was performed on cDNA synthesized from blood samples treated with these beads. We analysed 18 samples from 12 patients with a gastrointestinal disease, and 36 samples from ten patients with a colorectal cancer. None of the patients used as negative controls were found to contain epithelial cells in their blood as determined by cytokeratin 19 RT-PCR. By contrast, CD44 transcripts containing exon v6 were detected in nine out of the 18 samples tested (50%). For the colorectal cancer patients, six out of the seven samples (85.7%) that were cytokeratin 19-positive were CD44v6-negative, whereas ten samples out of the 29 not containing epithelial cells were CD44v6-positive (34.5%). This is probably due to the persistence of CD8+ leucocytes in the enriched preparations, as determined by PCR analysis of the CD8 α-chain. We conclude that detection of CD44v6 transcripts using a sensitive nested RT-PCR assay has no potential value to detect and characterize colorectal cancer micrometastases from blood, even following an initial enrichment step. © 2000 Cancer Research Campaign


MLH1, MSH2 and MSH6 mutation carriers.Lynch syndrome (LS) is associated with a high risk for colorectal cancer (CRC) and extracolonic malignancies, such as endometrial carcinoma (EC). The risk is dependent of the affected mismatch repair gene. The aim of the present study was to calculate the cumulative risk of LS related cancers in proven The studypopulation consisted out of 67 proven LS families. Clinical information including mutation status and tumour diagnosis was collected. Cumulative risks were calculated and compared using Kaplan Meier survival analysis.MSH6 mutation carriers, both males and females had the lowest risk for developing CRC at age 70 years, 54% and 30% respectively and the age of onset was delayed by 3-5 years in males. With respect to endometrial carcinoma, female MSH6 mutation carriers had the highest risk at age 70 years (61%) compared to MLH1 (25%) and MSH2 (49%). Also, the age of EC onset was delayed by 5-10 years in comparison with MLH1 and MSH2.MLH1, MSH2 and MSH6 mutations seem to cause distinguishable cancer risk profiles. Female MSH6 mutation carriers have a lower CRC risk and a higher risk for developing endometrial carcinoma. As a consequence, surveillance colonoscopy starting at age 30 years instead of 20-25 years is more suitable. Also, prophylactic hysterectomy may be more indicated in female MSH6 mutation carriers compared to MLH1 and MSH2 mutation carriers.Although the cumulative lifetime risk of LS related cancer is similar,  MLH1MLH1[MSH24MLH1[MSHMSH6[PMS2.MSH2 mutation families compared to MLH1 mutation families [MSH6 mutation families probably have a milder clinical phenotype with a later onset of both CRC and EC and clustering of endometrial carcinoma [PMS2 mutation families are largely unknown. One study reported that PMS2 mutation families have a milder phenotype compared to MLH1 and MSH2 families [The colorectal cancer risk in LS is dependent on sex and the MMR gene involved. The reported lifetime risk for colorectal cancer in the literature varies from 28-100% in males and 25-83% in females ,10-18. Tfamilies ,19. MSH6arcinoma . The risfamilies .MLH1 and MSH2 mutations, while studies evaluating MSH6 mutation families are sparse. The most efficient way to calculate the lifetime risks of CRC and EC in Lynch syndrome would be to calculate these risks based on a cohort of proven mutation carriers. Therefore, the aim of the present study was to calculate the cumulative lifetime risks for CRC and EC in Lynch syndrome using a cohort of proven MLH1, MSH2 and MSH6 mutation carriers.Unfortunately, the precise lifetime risk for CRC and endometrial carcinoma may be biased because the families selected in previous studies were mainly selected on basis of clustering of CRC or fulfilment of clinical criteria (Amsterdam II criteria). Furthermore, it was not always clear whether the affected subjects were proven mutation carriers. In addition, most studies have only evaluated lifetime risks for During the period 1994-2007, an MMR gene mutation was detected in 67 families who were counselled at the Department of Clinical Genetics of the Erasmus MC University Medical Center, because of a clinical suspicion for Lynch syndrome. Clinical data of family members including sex, age, mutation status, age at diagnosis of both LS-associated and other cancers were collected. LS-associated cancer included colorectal, endometrial, stomach, ovaries, upper uroepithelial tract, biliary tract, skin and brain cancer. Also, the site of the tumour, age at death and cause of death were collected. With consent of the patients or (in case the patient was deceased) of a close relative the cancer diagnosis was confirmed by pathology and/or medical reports. All pathology and medical reports were reviewed by the first author (DR) in order to confirm the diagnosis. If a subject reported the occurrence of cancer in the family and no pathology or medical report was available, the cancer was excluded from analysis. In addition, data regarding colonoscopic surveillance of affected and unaffected family members were collected.Only subjects with a proven MMR gene mutation were included in this study.http://www.hgvs.org. A variant was considered a mutation when leading to a predicted truncated protein or based on previously published data. Silent or missense variants which were previously unreported or of unclear status were labelled unclassified variants (UV) and not considered as an MMR gene mutation.Mutation analysis was performed by denaturing gradient gel electrophoresis, sequencing and multiplex ligation-dependent probe amplification (MRC-Holland kits P003 and P008). Mutation nomenclature was used according to international guidelines MLH1, MSH2 and MSH6 mutation carriers. Penetrance for age was calculated using the Kaplan Meier survival analysis method and included the 67 index cases. In case of multiple or recurrent colorectal carcinoma or endometrial adenocarcinoma, only the first diagnosis of either cancer was included in the analysis. The observation time for the different cancers was from birth until the date of first cancer diagnosis, death, date of hysterectomy  or the end of the study (31 December 2007). A p value below .05 was considered statistically significant.Data were submitted for statistical testing using the Statistical Package for the Social Sciences , version 12.0.1. Data are given as median and range or as mean with standard deviation when appropriate. The chi square test, Student's t test and log rank test were used to compare differences between MLH1 mutation, 20 (30%) with an MSH2 mutation and 21 (31%) with an MSH6 mutation. Of the 67 families, 46 (69%) met the Amsterdam II criteria. Mutation analysis was performed in 725 subjects (296 men and 429 women) and a mutation was identified in 246 subjects   mutation carriers developed another LS-associated cancer during their life  were detected with an One of the 69 mutation carriers had previously been diagnosed with EC and developed CRC while being under colonoscopic surveillance. The other 68 mutation carriers were included in a colonoscopic surveillance program after being diagnosed with colorectal cancer. These 68 subjects were treated surgically  for colorectal cancer and colonoscopic surveillance of the remaining colon was performed. Of the remaining 125 mutation carriers none developed colorectal cancer and in 23 (18%) adenomatous polyps had been detected and removed. The person-years of follow up was 1414 years and the mean follow up time of the subjects under colonoscopic surveillance was 7 ± 4 years.MLH1, 77% for MSH2 and 75% for MSH6 mutation carriers . This was due to the fact that before the age of 70 years the risk of developing any Lynch syndrome associated cancer in MSH6 carriers was lower compared to MLH1 or MSH2 mutation carriers  compared to MLH1 and MSH2 mutation carriers. However, the highest increase in risk in male MLH1 and MSH2 mutation carriers was observed between the ages of 40 to 50 years, while the risk in male MSH6 mutation carriers mostly increased between the ages of 50 to 60 years. Although the age related risks were not significant different between the three different MMR genes, there was a trend in male MLH1 and MSH2 mutation carriers to develop CRC at an earlier age than male MSH6 mutation carriers. The cumulative risks for CRC in females were lower compared to males, 57% for MLH1, 52% for MSH2 and 30% for MSH6 mutation carriers  compared to MLH1 and MSH2 mutation carriers.In Figure MSH6 mutation carriers (61%), while the cumulative risks for MLH1 and MSH2 mutation carriers were 25% and 49% respectively. However, the log rank test showed no significant difference in age related cumulative risk (p = 0.58) between MSH6 mutation carriers compared to MLH1 and MSH2 mutation carriers.For endometrial carcinoma, the highest cumulative risk was observed in the MSH6 mutation carriers  compared to MSH2 mutation carriers , but not significantly higher compared to MLH1 mutation carriers   with MLH1  and MSH2 . The median age of EC onset was significant higher in MSH6 mutation carriers  compared to MLH1 mutation carriers  and MSH2 mutation carriers . There were no significant differences in the age of onset of other LS-associated cancers between MLH1 , MSH2  and MSH6  mutation carriers .The median age of CRC onset in males was significantly higher in In this study, we evaluated 246 individuals from 67 families with a proven mismatch repair gene mutation to determine the cumulative lifetime risk of developing cancer. Previous studies have shown different lifetime risks for developing CRC in Lynch patients.MLH1 and MSH2 mutations. Thirty one percent of the families included in our study carried an MSH6 mutation. This frequency is higher than previously reported [MLH1, MSH2 and MSH6 families are sparse. A study evaluating the risk in 20 MSH6 families showed that colorectal cancer was less frequent and developed 10 years later in MSH6 compared to MLH1 and MSH2. In addition a significant higher lifetime risk of endometrial carcinoma of 71% in MSH6 mutation carriers with a later age of onset (54 years vs. 48 and 49 years for MLH1 and MSH2) was reported [MSH6 mutation families with 156 MLH1 and MSH2 mutation families confirmed the lower risk and later age of onset of CRC in MSH6 families [One of the first studies evaluating the lifetime risk reported a lifetime risk for CRC at age 75 years of 92% in males and 83% in females . Most lareported ,21-23. Sreported . A Germafamilies . These rfamilies .MLH1, MSH2 and MSH6 mutation carriers is similar, each mutated gene has a distinguishable cancer risk profile. In MSH6 mutation carriers the risk at age 70 years for developing CRC was the lowest in both male (54%) and female (30%) when compared to carriers of MLH1 and MSH2 mutations.Our study indicates that, however the cumulative risks of cancer at age 70 years in MSH6 and MSH2 mutation carriers also a significant difference in the age of CRC onset  was found and there was a trend in higher age of CRC onset between male MSH6 and MLH1 mutation carriers. For female mutation carriers, no significant differences were found in the mean age of onset of CRC. This can be explained by the fact that female MLH1 and MSH2 mutation carriers still developed CRC at an older age. The lower risk of CRC onset in female MSH6 mutation carriers under the age of 50 years raises the question whether colonoscopic surveillance guidelines in these subjects can be changed. Current guidelines advise to start with biennial colonoscopy surveillance from the age of 20-25 years [MSH6 mutation carrier with CRC was 34 years. Our data and the data from previous studies support that colonoscopic surveillance can be started at an age of 30 years in female MSH6 mutation carriers [Between male carriers .MLH1 mutation carriers. In MSH2 and MSH6 mutation carriers extracolonic cancers appear to contribute more to the similar cumulative lifetime risk of cancer in MLH1, MSH2 and MSH6 mutation carriers. A higher risk of extracolonic-LS-associated cancer was previously reported in MSH2 mutation carriers compared to MLH1 mutation carriers [MSH6 carriers have the highest endometrial cancer risk followed by MSH2 and MLH1 mutation carriers. Also, this risk increases sharply after the age of 50 years. In view of the disputable effect of endometrial carcinoma surveillance [MSH6 carriers aged 45 years or above prophylactic hysterectomy may be suggested in order to decrease the risk for developing endometrial carcinoma [MSH2 and MLH1 female mutation carriers this option may be more questionable. In MSH2 mutation carriers the risk of other extracolonic and extraendometrial cancers may reduce faith in and benefit of risk reducing surgery. In MLH1 mutation carriers the risk of endometrial cancer may not outweigh the disadvantages of surgery. In case of surgery for another cause, additional hysterectomy should be considered also in MLH1 en MSH2 mutation carriers.However our numbers are too small to draw definite conclusions, CRC seems to be the predominant cancer in carriers ,19. Unfocarriers ,26 our seillance ,28, in farcinoma . In MSH2MLH1, MSH2 and MSH6 mutation carriers.A strength of the present study was that the age related risks where calculated using proven mutation carriers. However, the age related risks might be somewhat lower since not all the unaffected individuals from proven mutation families opted for genetic testing and thus the total number of unaffected mutation carriers in the mutation families may be underestimated. In addition, individuals with a higher risk for mutation carriership, i.e. with an affected first degree relative, more often opt for genetic testing . This maMLH1, MSH2 and MSH6 mutation carriers are similar, each mutated gene has a distinguishable cancer risk profile. It underlines that female MSH6 mutation carriers have a distinct clinical phenotype with a lower CRC risk and a higher risk for developing endometrial carcinoma. Starting with biennial colonoscopic surveillance at an age of 30 years instead of an age of 20-25 years in female MSH6 mutation carriers is more suitable. Moreover, in female MSH6 mutation carriers prophylactic hysterectomy may be considered from an age of 45 years.In conclusion, the present study indicates that, although the cumulative risks at age 70 years of LS related cancer in MSH6 mutation carriers have a lower CRC risk and a higher risk for developing endometrial carcinoma. Starting with biennial colonoscopic surveillance at an age of 30 years in female MSH6 mutation carriers is more suitable and prophylactic hysterectomy may be considered from an age of 45 years.The present study indicates that each mutated MMR gene has a distinguishable cancer risk profile. Female CRC: colorectal cancer; EC: endometrial cancer; LS: Lynch syndrome; MMR: mismatch repair; UV: unclassified variant.The authors declare that they have no competing interests.DR participated in the data collection, performed the statistical analyses and helped to draft the manuscript. AW conceived of the study and participated in the data collection. ML helped to draft the manuscript. DD participated in the data collection. CT participated in the data collection. ES participated in the design of the study and assisted in the statistical analysis. EK helped to draft the manuscript. All authors read and approved the final manuscript.


P <0·001) higher than in a healthy control group, but after local therapy the concentration was significantly lower than before treatment (P <0·01). The concentration of conjugated 3OHA was nearly constant in the three groups. It was concluded that other factors than a genetic determined abnormality might be operating in bladder cancer patients which could lead to an abnormal concentration of 3OHA in their urine.The basal concentration of the tryptophan metabolite 3-hydroxyanthranilic acid (3OHA), which has carcinogenic properties, was measured with an enzymatic method of determination which allowed separate measurement of free and conjugated 3OHA. The concentration of free 3OHA in untreated bladder cancer patients was significantly (


We screened the 185delAG and 5382insC (BRCA1) and the 6174delT (BRCA2) mutation in 298 Spanish women with breast cancer. Two women (one with Sephardic ancestors) presented the 185delAG mutation and the same haplotype reported in Ashkenazim with this mutation. This suggests a common origin of the 185delAG in both Sephardic and Ashkenazi populations. © 1999 Cancer Research Campaign


Circulating tumor cells (CTC) and disseminated tumor cells (DTC) are thought to be responsible for metastasis, so the detection of CTC may serve as individual prognostic factor in patients suffering from colorectal cancer. Therefore, a series of immunomagnetic enrichment methods for CTC have been developed using a variety of monoclonal antibodies against the Epithelial Cell Adhesion Molecule (EpCAM). However, it remains unclear whether all commercially available EpCAM antibodies show the same sensitivity and specificity. Furthermore, it remains unclear which method of sample preparation and cell extraction is most suitable for immunomagnetic enrichment and detection of CTC. In this study, we aimed to investigate whether the detection of CTC by a cytokeratin 20 reverse transcriptase-polymerase chain reaction (CK20 RT-PCR) may be influenced by the use of various Epithelial Cell Adhesion Molecule (EpCAM) antibodies for immunomagnetic isolation of CTC.Using both EpCAM antibodies (mAb BerEP4 and mAb KS1/4) for immunomagnetic enrichment in blood samples of 39 patients with colorectal cancer we found heterogenous results in each patient with regard to tumor cell detection. In the tumor cell spiking experiments with whole blood samples the sensitivity of the CK 20 RT-PCR assay was higher using immunomagnetic beads coated with mAb KS1/4 compared to precoated mAb BerEP4 Dynabeads. Extraction of MNC fraction with Ficoll gradient centrifugation prior to immunomagnetic enrichment resulted in a higher sensitivity of the CK 20 RT-PCR assay.We concluded that isolation and detection of CTC with immunomagnetic enrichment methods is critically dependent on the used EpCAM clone. Further studies with a larger number of patients should clarify if the enrichment protocol influences the prognostic value of the tumor cell detection protocol. Detection of circulating tumor cells (CTC) in blood and disseminated tumor cells (DTC) in the bone marrow and/or lymph nodes, which are thought to be responsible for metastases, may allow a better prediction of the individual prognosis of patients with colorectal cancer -3. RecenIn this study, we aimed to compare two different specific antibodies against the epitope in the EGF-like domain I of EpCAM for immunomagnetic enrichment and subsequent detection of CTC in CRC patients. We used commercially available immunomagnetic beads coated with mAb BerEP4  and magnBoth whole blood and MNC fractions of five healthy donors were tested regarding the specificity of cell extraction and enrichment protocols with immunomagnetic beads coated with BerEP4 and KS1/4. No CK20 signal was observed in all examined blood samples of healthy donors, demonstrating the specificity of the used assays.4 HT29 cells could be detected in 5 ml whole blood using the BerEP4 mAb whereas 103 HT29 cells could be detected in the same volume using the KS1/4 mAb.In the tumor cell spiking experiments with whole blood samples the sensitivity of the CK20 RT-PCR assay was higher using immunomagnetic beads coated with mAb KS1/4 compared to precoated mAb BerEP4 Dynabeads. In serial dilution assays, a minimum number of 103 HT29 cells spiked in 5 ml blood (200 cells/ml) could be detected after isolation of the MNC fraction using the mAb BerEP4 Dynabeads, whereas 102 HT29 cells spiked in 5 ml blood (20 cells/ml) were detected using the mAb KS1/4 coated beads.Extraction of MNC fraction with Ficoll gradient centrifugation prior to immunomagnetic enrichment of blood samples spiked with HT29 cells resulted in a higher sensitivity of the CK20 RT-PCR assay. 10Blood spiking experiments were repeated several times to confirm the above mentioned results.The observed higher sensitivity of tumor cell detection after isolation of the MNC fraction prior to immunomagnetic CTC enrichment prompted us to generally use Ficoll gradient centrifugation before further immunomagnetic enrichment and detection of CTC in the blood of CRC patients.Median age of included CRC patients was 63 years (range 27 - 90) with 16 females and 23 males. Eighteen of 39 (46%) patients presented with metastatic disease to the liver and were classified as UICC stage IV; 7 patients were UICC stage III, and 7 patients stage UICC II and I. Clinical data of the patients are shown in Table Using two different antibodies (mAb BerEP4 and mAb KS1/4) for immunomagnetic enrichment, CTC were detected in 11 of 39 28%) patients with CRC. Among these, immunomagnetic enrichment with mAb BerEP4 beads accounted for 6 CK20 positive patients. Immunomagnetic enrichment using mAb KS1/4 beads showed 5 CK20 positive patients. Interestingly, there were no blood samples being CK 20 positive for both used antibodies (Table 8% patienCK20 PCR transcripts were detected in 3 of 18 (17%) blood samples from UICC stage IV patients after immunomagnetic enrichment with mAb KS1/4 coated beads. When mAb BerEP4 beads were used, we also observed positive CK20 products in 3 of 18 (17%) UICC stage IV patients. Among the 7 patients with UICC stage III, 3 patients showed CK20 positive samples after enrichment of CTC with BerEP4 beads only. Positive CK20 signals were observed in 2 of 7 patients with UICC stage I after enrichment with KS1/4 coated beads only  after MNC gradient enrichment.To our knowledge, this is the first study reporting the use of mAb KS1/4 coated beads to isolate CTC from CRC patients. The sensitivity of the KS1/4 coated bead system was evaluated by HT29 tumor cell dilution experiments and reproducibly allowed the detection of about 10Our study also confirmed the study from Guo et al. which showed better results for MNC population extraction prior to immunomagnetic isolation of tumor cells than the immunomagnetic isolation of circulating tumor cells from whole blood . Guo et BerEP4 and KS1/4 are high affinity and specific mAb frequently used to detect EpCAM positive cells. Interestingly, we noted that CK20 RT-PCR products were commonly not found in both samples when analyzing blood of the same patient after enrichment with either mAb BerEP4 or mAb KS/4. Other authors have already described heterogeneity in reactivity of EpCAM specific antibodies. Balzar et al. suggested that different conformational states of the cell surface EpCAM protein might hide some epitopes leading to subpopulations of EpCAM and thus heterogeneous affinity .Successful CTC enrichment depends on the level of EpCAM expression in the target cell . FurtherThe dissimilar capture of CTC by BerEP4 and KS1/4 mAbs in our analysis might be explained by various expression of the EpCAM molecule among the examined patients. Nevertheless, our KS1/4 system was able to retrieve 10 fold more CTC compared to the BerEP4 system. The use of different EpCAM clones might enhance the detection rate of CTC. Additional investigations to assess the variations of EpCAM molecule expression among different patients will be of critical importance to identify a panel of suitable mAbs which could be used for efficient and reliable CTC isolation.Our results also demonstrate that Ficoll-gradient isolation is a decisive step prior to the immunomagnetic enrichment of CTC from peripheral blood.Our study for the first time shows that isolation and detection of CTC with immunomagnetic enrichment methods is critically dependent on the used EpCAM clone. Further analysis regarding the clinical importance of heterogenous expression of the EpCAM molecule in CTC of CRC patients is urgently needed.®, Sarstedt, Germany). To avoid epithelial cell contamination from skin puncture, the first 5 ml of peripheral blood were discarded. After collection, blood samples were immediately processed for further experiments.For blood spiking experiments and for testing the specificity of the various extraction and enrichment protocols peripheral blood samples were drawn from the antecubital vein of 5 healthy donors and collected in EDTA-coated tubes (S-Monovetteth edition) [Five ml blood samples were obtained after induction of general anesthesia (and before start of the operation) through a central venous line from 38 CRC patients (UICC stage I-IV) undergoing surgical therapy at the Department of Surgery, University of Heidelberg, Germany. Patients with histopathologically confirmed CRC were staged according to the classification of the UICC (6edition) . The stu6, 105, 104, 103, 102, 10 and 0 HT29 cells per 5 ml whole blood.For cell spiking experiments and the examination of different cell extraction methods, EpCAM and CK20 positive cells from the human colon cancer cell line HT29 were serially diluted in 5 ml blood samples taken from five different healthy donors. Dilutions performed were: 102 atmosphere. Collection of cells was performed with help of Trypsin-EDTA  and centrifugation at room temperature for 3 minutes at 300 g. Cells were then counted with a hemacytometer and viability was confirmed by Trypan blue stain.The human colon cancer cell line HT29 expressing EpCAM and Cytokeratin 20 (CK20) was purchased from American Type Cell Culture (Austria Branch). Cells were cultured in RPMI-1640 Medium with L-glutamine and HEPES  supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin , and 10% fetal bovine serum  in plastic flasks at 37°C in a 5%CO®, Linaris, Germany density 1.077) and covered with 10 ml phosphate buffered saline solution . The samples were spun in a centrifuge at 4°C for 30 minutes at 300 g without brake. Concentrated MNCs were harvested from the interface with the help of a disposable pipette. The isolated cells were washed once in PBS, spun in a centrifuge for 10 minutes at 300 g and resuspended in 1 ml PBS. The MNCs were counted with a hemacytometer and then resuspended at 107 cells/ml in PBS.The mononuclear cell (MNC) population was extracted according to the following protocol: 5 ml peripheral blood samples were carefully layered over a 15 ml Ficoll gradient (FicoLite-HThe unspiked and spiked (with HT29 cells) blood samples (5 ml blood for each experiment) of healthy volunteers were processed using two different cell extraction protocols followed by immunomagnetic enrichment using the device of Dynal MPC-L :7 beads per ml blood) using either Dynabeads Epithelial Enrich  coated with mAb BerEP4 or alternatively, Pan Mouse IgG beads  coated (1 μg antibody/107 beads) with the mAb KS1/4 . The samples were then placed in a roller at 4°C for rosetting to occur and after 30 minutes the tubes were placed in a magnetic device for 3 minutes; the blood supernatant was carefully removed and, while the tubes were still on the magnet, rosettes were washed three times with cold PBS.1. Whole blood samples underwent directly an immunomagnetic enrichment  was performed prior to further immunomagnetic enrichment. Immunomagnetic enrichment using the two different EpCAM antibodies was performed as described above.CK20 transcripts were detected after immunomagnetic enrichment of tumor cells either derived from blood samples spiked with HT29 cells or from blood samples drawn from CRC patients.For the detection of tumor cells a CK 20 nested RT-PCR was performed as previously shown ,13. In bFor the first PCR cDNA was subjected to amplification of CK20 with primers 1.for (ATGGATTTCAGTCGCAGA) and 558.rev (ATGTAGGGTTAGGTCATCAAAG) in 35 amplification rounds performed at 93°C for 51 seconds, 60°C for 63 seconds, and 72°C for 42 seconds, with a final extension step at 72°C for 10 minutes. The nested PCR was performed with 8 μl PCR product of the first PCR with primer 139.for (TCCAACTCCAGACACACGGTGAACTATG) and 429.rev (CAGGACACACCGAGCATTTT GCAG) under amplification conditions as following: 93°C for 51 seconds and 72°C for 81 seconds in 35 amplification rounds. PCR products were analyzed by electrophoresis on 2% agarose gels. RNA quality and performance of reverse transcription of the analyzed samples was confirmed by RT-PCR amplification of GAPDH transcripts.DA, LG, and AC performed the experiments and contributed to clinical data collection and manuscript preparation. NR, MWB, and JW contributed to the conduction of the study and were involved in critical review and revision of the manuscript. MK designed the study and was responsible for manuscript preparation and revision. All authors read and approved the final manuscript


To determine resistance of highly pathogenic avian influenza (H5N1) virus to chlorination, we exposed allantoic fluid containing 2 virus strains to chlorinated buffer at pH 7 and 8, at 5°C. Free chlorine concentrations typically used in drinking water treatment are sufficient to inactivate the virus by >3 orders of magnitude. Growing concerns about the public health threat posed by highly pathogenic avian influenza (HPAI) subtype H5N1 has prompted interest in evaluating environmental control measures for this virus. The World Health Organization has noted that more information is needed on the effectiveness of inactivation of subtype H5N1 in water  virus were used in this study /mL (oC under 5% CO2 for 96 h and examined by light microscopy for cytopathic effect (CPE). Culture plates were stained with 1% (w/v) crystal violet in 10% (v/v) neutral-buffered formalin for further examination. Failure to produce CPE indicated that the virus was not capable of infecting the cells. The neutralized buffer control without virus did not cause CPE. All experiments were conducted in duplicate under Biosafety Level 3 agricultural conditions.The infectivity of the samples was quantified by using microtiter endpoint titration  were used to determine the rate of inactivation for the 2 pH levels. Ct values are commonly used to make disinfection recommendations for water treatment and provide a means for comparing biocidal activity for various microorganisms  required to achieve 1, 2, and 3 orders of magnitude inactivation for both strains at the 2 pH levels. Covariance analysis of the decay coefficients indicated no significant difference in the inactivation of the 2 virus strains at pH 8.0 (p = 0.10). Rapid inactivation at pH 7.0 did not allow for statistical evaluation.The results of the chlorination experiments  represenCt values of 6 and 8 mg-min/L to achieve enteric virus inactivation of 3 and 4 orders of magnitude, respectively  in the water environment. The information on chlorine disinfection presented here should be helpful for developing risk management procedures regarding the role of water in the transmission of the virus to humans and poultry.The results of this study confirm that avian influenza (H5N1) is readily inactivated by chlorination. Although the viral inoculum exerted a considerable initial chlorine demand, the maintenance of a free chlorine residual (0.52–1.08 mg/L) was sufficient to inactivate the virus by >3 orders of magnitude within an exposure time of 1 minute. Chlorine demand would also be anticipated when the virus is associated with fecal material. These findings indicate that the ability to compensate for an initial chlorine demand followed by exposure to a relatively low level of free chlorine for a short time is sufficient to inactivate the virus by chlorination. For drinking water disinfection at conditions similar to those used in this study, the US Environmental Protection Agency specifies free chlorine


The purpose of this study was to evaluate the efficacy of HDR brachytherapy for primary or recurrent vaginal cancer.Between the years 2000 to 2006, 18 patients with primary or recurrent vaginal cancer were treated with brachytherapy (HDRB). Six patients had primary vaginal cancer (stage II to IVA) while 12 were treated for isolated vaginal recurrence . Five patients had previous pelvic radiation therapy. All except one patient received external beam radiation therapy to a median dose of 45 Gy (range 31.2–55.8 Gy). The HDRB was intracavitary using a vaginal cylinder in 5 patients and interstitial using a modified Syed-Nesblett template in 13 patients. The dose of interstitial brachytherapy was 18.75 Gy in 5 fractions delivered twice daily. The median follow-up was 18 months (range 6–66 months).Complete response (CR) was achieved in all but one patient (94%). Of these 17 patients achieving a CR, 1 had local recurrence and 3 had systemic recurrence at a median time of 6 months (range 6–22 months). The 2-year actuarial local control and cause-specific survival for the entire group were 88% and 82.5%, respectively. In subset analysis, the crude local control was 100% for primary vaginal cancer, 100% for the group with recurrence without any prior radiation and 67% for group with recurrence and prior radiation therapy. Two patients had late grade 3 or higher morbidity . Both these patients had prior radiation therapy.Our small series suggests that HDRB is efficacious for primary or recurrent vaginal cancer. Patients treated with primary disease and those with recurrent disease without prior irradiation have the greatest benefit from HDRB in this setting. The salvage rate for patients with prior radiation therapy is lower with a higher risk of significant complications. Additional patients and follow-up are ongoing to determine the long-term efficacy of this approach. Primary or recurrent vaginal carcinoma is an uncommon tumor . The iniBrachytherapy has been shown to be an important component of treatment in these patients. Treatment selection can be adapted to account for stage and location of the tumor. It can be done with either intracavitary or interstitial approach. The majority of published studies with interstitial brachytherapy have reported data using low-dose-rate brachytherapy (LDRB) -9. ThereThe purpose of this study was to evaluate the efficacy and toxicities of HDRB for primary or recurrent vaginal cancer.Between January 2000 and December 2006, 18 patients with primary or recurrent vaginal cancer were treated with HDRB. The median age was 69 yrs (range 43–88 yrs). Six patients had primary vaginal cancer . Twelve patients were treated for isolated vaginal recurrence . The stage and grade (G) of endometrial cancer were IB G1-1 patient, IB G2-2 patients, IC G3-2 patient, IIA G 3 – 1 patient and IIB G2 – 1 patient. The median time to recurrence for all patients was 12 months (range 3 months – 18 years). Six of these patients had prior pelvic radiation therapy. The type of previous radiation was EBRT alone 1 patient, EBRT plus brachytherapy 3 patients and brachytherapy alone 2 patients. The median time since previous radiation to recurrence was 4 years (6 months – 18 years). The recurrence was marginal (within 2 cm of previous field) in 2 patients and within the previous field in 4 patients. The sites of disease were proximal vagina 9 patients, distal vagina 6 patients and diffuse disease in 3 patients.All except one patient received a combination EBRT and HDRB. The median dose of EBRT was 45 Gy (range 31.2–55.8 Gy) at 1.8 to 2 Gy per fraction. The technique of EBRT was 3D conformal in 9 patients and IMRT in 8 patients. Five out of six patents with vaginal cancer also had concurrent weekly cisplatinum at 40 mg/m2 along with EBRT.The HDRB was intracavitary brachytherapy for superficially invasive tumors (less than 5 mm of invasion), while interstitial brachytherapy was used for more deeply invasive tumors greater than 5 mm. Five patients had intracavitary brachytherapy utilizing Delclos Vaginal Applicators system. Two of these patients had shielded vaginal applicators to protect the uninvolved vaginal mucosa. Orthogonal X-ray films were obtained for planning and verification of applicator placement. The median dose for intracavitary brachytherapy was 20 Gy 12 – 20 Gy) in 3–5 fractions prescribed at 0.5 cm from the surface of the applicator. One of these patients had intracavitary brachytherapy only because of previous radiation therapy with EBRT plus HDR brachytherapy. Interstitial brachytherapy using a modified Syed-Nesblett template was performed in 13 patients. We have previously described this technique [2 – 20 GyThe biologically equivalent dose (BED) in terms of equivalent doses given at 2 Gy per day (EQ2) was calculated using the LQ equation . The α/βThe median follow-up was 18 months (range 6–66 months) for the entire group. Complete response (CR) was achieved in all but one patient (94%). This one patient with previous radiation therapy had recurrent papillary serous endometrial cancer and had partial regression of the tumor. Of the 17 patients achieving a CR, 1 had local recurrence and 3 had systemic recurrence at a median time of 6 months (range 6–22 months). The one local recurrence was in the patient treated with previous adjuvant radiation therapy for endometrioid adenocarcinoma. Two patients have died of disease at 12 and 18 months, respectively. Both patients had recurrent endometrial cancer with papillary serous and carcinosarcoma histology, respectively. Persistence of local disease was noted in one of these patients. The 2-year actuarial local control, cause-specific and overall survival for the entire group was 88%, 82.5% and 78%, respectively. In subset analysis, the crude local control was 6/6 (100%) for primary vaginal cancer, 6/6 (100%) for the group with recurrence without any prior radiation and 4/6 (67%) for group with recurrence and any prior radiation therapy.No grade 3 or worse early toxicity was observed. Two patients had late grade 3 or 4 morbidity. One patient had rectovaginal fistula 2 years after radiation therapy and other patient had chronic vaginal ulcer with significant narrowing and shortening of vagina . Both these patients had proximal vaginal disease and had prior radiation therapy and their EQ 2 for total doses were 142.98 Gy and 154 Gy, respectively. The cumulative doses were highest in these two patients. No other significant grade 3 or higher morbidity was noted.Radiotherapy is the main therapeutic modality in the management of primary or recurrent vaginal cancer. Brachytherapy remains integral part of definitive radiation therapy for these patients -9. The pet al. on 22 patients with recurrent endometrial cancer reported local tumor control rate and the 5-year disease-specific survival rate of 100% and 96%, respectively [et al. on 20 patients, 10-year local control rate and disease-free survival rates were 74% and 46%, respectively [In a review of the literature regarding salvage treatment options for vaginal recurrence, only two studies have examined the efficacy of HDRB for treatment of vaginal recurrences ,15. The ectively . Similarectively .Neblett template implantation procedure delivering 5 fractions twice a day for a total HDRB dose of 18.75 Gy over 48 – 56 hours. Because of lack of published data, the American Brachytherapy Society (ABS) did not make any recommendation on HDRB interstitial brachytherapy dose and fractionation schedule for vaginal recurrences and preferred LDRB as the technique for interstitial brachytherapy [Interstitial brachytherapy has been traditionally performed using LDRB techniques in this setting. Our technique involved one Syed-ytherapy . Our treet al. first reported on 19 patients treated with the combination of intracavitary and interstitial brachytherapy [n = 26/86), whereas patients with locally advanced disease (stages II-IV) received HDR brachytherapy combined with external beam therapy (n = 55/86). The prescribed dose per fraction varied from 5 Gy to 8 Gy, with a mean dose of 7 Gy. In this large series only 8 patients had interstitial brachytherapy. The 5-year recurrence-free survival were 100%, 77%, 50%, 23%, and 0% for stage 0, I, II, III and IV respectively. Chronic grade 1–4 side effects were observed in ≤ 2%  and 1%–6% (vagina). Our series only had 6 patients of primary vaginal cancer and all had interstitial brachytherapy with 2-year local control of 100%. The toxicity profile was favorable with no grade 3 or higher toxicities.Similarly, there are only few published studies on HDRB for primary vaginal cancer. Kusher, ytherapy . The medytherapy . Early set al. noted an increasing risk of local recurrences in patients who received <55 Gy when compared with those receiving >55 Gy (53% vs. 17%) [et al. reported local failures in 25%, 33% and 62% of patients for the administered dose of >75 Gy, 60–75 Gy and <60 Gy respectively [In LDRB literature for vaginal cancer, an inverse relationship between the total dose and rates of local recurrences have been reported by several authors ,18. Chylvs. 17%) . Similarectively . With a Notwithstanding, our small series with preliminary results shows that HDRB is efficacious for primary or recurrent vaginal cancer. The fractionation schedule used was well tolerated with a low incidence of acute or later toxicities. Additional patients and follow-up are ongoing to determine the long-term efficacy of this approach. The limitation of our retrospective study is small size with limited follow up and heterogeneous patient population evaluated (inclusion of both primary and recurrent disease). The incidence of primary or recurrent vaginal cancer is low so that it is difficult for a single institution to have a large series. That was the rationale to combine heterogeneous disease together to see the efficacy and toxicities of this approach. As HDR equipment is widely available, there are more institution doing HDR interstitial brachytherapy. We may need to consider multi-institutional pooled analysis similar to the LDR experience  to see tSB – Took part in design and implementation of study, drafted manuscript, performed statistical analysis. DEH – Lead in drafting manuscript. RM – Helped in data collection. RPE – Participated in study design and patient selection. JLK – Participated in study design and patient selection. PS – Conceived the study, participated with design and coordinated/helped with patient selection. All authors read and approved the final manuscript.


Sir,AKT1 in about 2% of endometrial cancer patients (PTEN and PIK3CA (PIK3CA (PTEN (The activating E17K mutations recently discovered in the pleckstrin homology domain of AKT2 (D399N) and the regulatory domain of AKT3 (E438D)) were previously reported in a sequencing screen of 123 genes in 41 primary endometrial cancers  in two additional samples. We validated these mutations as somatic by mass spectrometric genotyping of the tumour and matched normal DNA, after an independent PCR amplification. We also found a novel candidate mutation in the pleckstrin homology domain of AKT2 (D32H), which we could not validate because of insufficient DNA. All these mutations occurred in cancers of the endometrioid subtype that had no signs of metastasis either at primary treatment or during follow-up. Taken together, we find that 5 out of 41 endometrial cancers have mutations in AKT family members for a 12% rate.We found an additional four mutations in AKT family members . Two of PTEN, one of which also had amplification of and a mutation in PIK3CA (AKT1 E17K mutation is not associated with either PTEN or PIK3CA genomic alteration. It is therefore possible that these AKT family mutations have different functional effects from mutations of PTEN and PIK3CA. Given the importance of the PI3 kinase pathway in endometrial cancer oncogenesis (Confirmation that these novel mutations activate the PI3 kinase pathway awaits their functional characterisation. Notably, all the AKT family member mutations found in our data occur at residues conserved across multiple species see . Howevern PIK3CA ; the AKTogenesis , the fun