Many plasma membrane transporters in yeast are endocytosed in response to excess substrate or certain stresses and degraded in the vacuole. Endocytosis invariably requires ubiquitination by the HECT domain ligase Rsp5. In the cases of the manganese transporter Smf1 and the amino acid transporters Can1, Lyp1 and Mup1 it has been shown that ubiquitination is mediated by arrestin-like adaptor proteins that bind to Rsp5 and recognize specific transporters. As yeast contains a large family of arrestins, this has been suggested as a general model for transporter regulation; however, analysis is complicated by redundancy amongst the arrestins. We have tested this model by removing all the arrestins and examining the requirements for endocytosis of four more transporters, Itr1 (inositol), Hxt6 (glucose), Fur4 (uracil) and Tat2 (tryptophan). This reveals functions for the arrestins Art5/Ygr068c and Art4/Rod1, and additional roles for Art1/Ldb19, Art2/Ecm21 and Art8/Csr2. It also reveals functional redundancy between arrestins and the arrestin-like adaptors Bul1 and Bul2. In addition, we show that delivery to the vacuole often requires multiple additional ubiquitin ligases or adaptors, including the RING domain ligase Pib1, and the adaptors Bsd2, Ear1 and Ssh4, some acting redundantly. We discuss the similarities and differences in the requirements for regulation of different transporters. Many yeast plasma membrane transporters are removed from the cell surface in response to excess substrate, nutrients or stress. It is well established that their endocytosis and subsequent vacuolar degradation are controlled by ubiquitination, and in every case tested, ubiquitination requires the HECT domain E3 ligase Rsp5 How a single ubiquitin ligase recognizes a wide variety of substrates is now beginning to be understood. Rsp5 contains three WW domains, which recognize PY motifs with the typical sequence PPXY or LPXY. Several adaptor proteins containing such motifs have been shown to facilitate the ubiquitination of particular proteins or sets of proteins  and to the four transporters mentioned above. Several other permeases appeared unaffected in arrestin mutant strains, possibly because of functional redundancy of the arrestins. We therefore sought to extend the findings to other proteins, to test the generality of the model and see whether a single mechanism can account for the behaviour of all regulated transporters.Here we identify adaptors required for regulated degradation of the inositol permease Itr1, hexose transporter Hxt6, uracil permease Fur4, and tryptophan transporter Tat2. We show that Bul1 and Bul2 have functions related to those of the arrestins and may share structural features with them. We also show that the Rsp5 adaptor Bsd2 and the RING domain ligase Pib1 provide an additional redundant function required for several transporters to negotiate the multivesicular body pathway. The results reveal common themes but also considerable variation in the requirements for individual proteins to be endocytosed to the vacuole.Our previous studies suggested considerable functional redundancy amongst the yeast arrestins. Our strategy therefore was to eliminate all of them, test the effect on a given transporter, then add back plasmids encoding single arrestins to see which were best at restoring endocytosis. This strategy required all of the relevant arrestins to be identified.Yeast contains eight proteins that have clear arrestin sequence signatures and show readily detectable homology to each other, namely Art2/Ecm21, Art8/Csr2, Art4/Rod1, Art7/Rog3, Art6/Aly1, Art3/Aly2, Art5/Ygr068c and Art9/Rim8 We identified an additional protein, Ylr392c, which also shows a match to the pfam arrestin_N domain  yet is d9-arrestin mutant, lacking all the PY-containing arrestins, which was healthier. To allow studies of the endocytosis of the metal transporter Smf1 this strain also lacked Bsd2, an adaptor that in normal medium causes newly synthesized Smf1 to be diverted from the Golgi to the vacuole. We refer to this strain as the 9-arrestin mutant.We constructed a yeast strain in which all 10 arrestins were deleted. Although viable, this strain grew more slowly than wild-type. This was at least partly because of the loss of Rim8, which is required for robust growth under a variety of conditions. Because Rim8 seems to have a distinct function, for subsequent endocytic studies we primarily used a The inositol transporter Itr1 is endocytosed in response to exogenous inositol rsp5 mutant, and also in the 9-arrestin mutant strain  GFP in response to either uracil or cycloheximide was reduced in the BUL1 was deleted in addition to the arrestins  machinery bsd2 cells (our unpublished observations), suggesting that these lysines may be sites of Pib1 and/or Ear1/Ssh4 dependent ubiquitination.It seems most likely that the additional ubiquitination occurs mainly in endosomes, as Bsd2 and Ear1/Ssh4 are membrane proteins that are themselves substrates for Rsp5 and are targeted to the vacuole apparently without reaching the plasma membrane One obvious question is how these disparate proteins recognize their substrates. Ear1 and Ssh4 contain SPRY domains, which may mediate protein–protein interactions, but they appear to act on every protein tested, from the vacuolar enzyme Phm5, with a single transmembrane domain and a tiny cytoplasmic domain, to large multispanning transporters such as Fur4 and Smf1 erg6 mutant, defective in ergosterol synthesis, the stable compartments do not form These considerations suggest a view of transporter regulation in which specificity is provided by arrestin interactions which promote initial internalization, and this is followed by relatively non-specific further ubiquitination in endosomes. This places great importance on the initial endocytic step, and it is worth considering what else might influence this. Notably, it has been shown that Can1, Tat2 and Fur4 are localized within small highly stable ergosterol-rich compartments in the plasma membrane whose formation depends on specific proteins, and that this slows their endocytosis by sequestering them from the sites at which this happens It seems that there are considerable differences in the way that the presence of individual transporters at the plasma membrane is controlled, though arrestins provide a common theme and may in some cases mediate specific regulatory mechanisms. Further exploration will no doubt reveal additional features, and may assign roles to the four arrestins whose functions are currently unknown. The arrestin-deficient strains we have developed should provide useful tools for future study.MATa his3-Δ1 leu2-Δ0 met15-Δ0 ura3-Δ0) or BY4742 (MATαhis3-Δ1 leu2-Δ0 lys2-Δ0 ura3-Δ0) and are listed in Schizosaccharomyces pombe HIS5 gene flanked by loxP sites, then using transient expression of the cre protein from a plasmid to remove the HIS5 marker.All yeast strains were derivatives of BY4741 . Bsd2 was expressed from either its own promoter (with Fur4 and Hxt6) or the TPI promoter (with Tat2 and Itr1). Strains were grown in appropriate selective media to maintain the plasmids. Growth rates in Plasmids were based on the YCplac111 600 less than 0.3). For Smf1, synthetic medium with glucose and appropriate supplements was used, and cadmium treatment was performed as described previously m myo-inositol. For Hxt6, cells were grown in YEP medium (1% yeast extract and 2% peptone) with 2% raffinose to an OD600 of approximately 1, then endocytosis induced either by adding 100 µg/mL cycloheximide, or by transferring to medium containing 0.17% yeast nitrogen base without ammonium sulphate or amino acids but with 5% glucose m uracil. For Tat2, the same protocol was used as for Fur4, except that cells were grown on synthetic medium lacking tryptophan and leucine, and endocytosis was induced by adding 1.5 mg/mL tryptophan.For fluorescence experiments cells were usually grown to exponential phase (ODImmunoblotting was performed on total cell extracts prepared using the alkaline lysis method Fluorescence imaging was performed on live cells in growth medium using a Zeiss LSM510 confocal microscope as previously described


HER2) fluorescence in situ hybridization (FISH) is a quantitative assay for selecting breast cancer patients for trastuzumab therapy. However, current HER2 FISH procedures are labor intensive, manual methods that require skilled technologists and specialized fluorescence microscopy. Furthermore, FISH slides cannot be archived for long term storage and review. Our objective was to develop an automated brightfield double in situ hybridization (BDISH) application for HER2 gene and chromosome 17 centromere (CEN 17) and test the assay performance with dual color HER2 FISH evaluated breast carcinomas.Human epidermal growth factor receptor 2  and the fast red and naphthol phosphate reaction with alkaline phosphatise (AP), respectively. The BDISH specificity was optimized with formalin-fixed, paraffin-embedded xenograft tumors, MCF7 (non-amplified HER2 gene) and BT-474 (amplified HER2 gene). Then, the BDISH performance was evaluated with 94 routinely processed breast cancer tissues. Interpretation of HER2 and CEN 17 BDISH slides was conducted by 4 observers using a conventional brightfield microscope without oil immersion objectives.The BDISH assay was developed with the nick translated dinitrophenyl (DNP)-labeled HER2 and CEN 17 ISH demonstrated both DNA targets in the same cells. HER2 signals were visualized as discrete black metallic silver dots while CEN 17 signals were detected as slightly larger red dots. Our study demonstrated a high consensus concordance between HER2 FISH and BDISH results of clinical breast carcinoma cases based on the historical scoring method  and the ASCO/CAP scoring method with the FISH equivocal cases  and without the FISH equivocal cases .Sequential hybridization and signal detection for HER2 and CEN 17 targets were successfully developed and it might be able to replace manual two-color HER2 FISH methods. The application also has the potential to be used for other gene targets. The use of BDISH technology allows the simultaneous analyses of two DNA targets within the context of tissue morphological observation.Automated BDISH applications for  HER2) oncogene, located on the long arm of chromosome 17 (17q12-q21), is over-expressed or amplified in approximately 20% of breast carcinoma cases [HER2 status in breast cancer is used as a prognostic factor, a predictive factor, and a therapy selection factor [®; Genentech), which is an FDA approved drug for use as monotherapy or combined chemotherapy for treatment of breast cancer patients with amplified HER2 status. Trastuzumab adjuvant treatment for early HER2 positive breast cancer is effective for improving patient survival and cost-effectiveness analyses of such treatment have shown acceptable ratios [HER2 gene over-expression associated with anthracycline treatment [The human epidermal growth factor receptor 2  analyses for detecting HER2 gene amplification and semi-quantitative HER2 immunohistochemistry (IHC) analyses for detecting over-expressed HER2 protein are performed to determine the HER2 status of breast cancer patients. The optimal scoring method for determination of HER2 gene status is the use of chromosome 17 centromere (CEN 17) enumeration for calculating the HER2/CEN 17 ratio [HER2 gene amplification have better prognosis compared to patients with HER2 gene amplification [HER2 and CEN 17 targets is recommended especially for borderline IHC cases [HER2 FISH assays beyond the requirement for a specialized fluorescence microscope and the difficulty of preserving FISH signal during a long term storage. For example, HER2 FISH testing has exhibited a higher assay failure rate in the hands of some investigators when compared to HER2 IHC testing (5% vs. 0.08%), the FISH assay procedure time is longer than the IHC assay (36 hours vs. 4 hours), and the FISH interpretation time is longer than IHC interpretation time (7 minutes vs. 45 seconds) [HER2 proficiency testing study showed that there was 20% (4 out of 20 samples) discordance with HER2 FISH testing among 5 experienced laboratories [Quantitative 17 ratio . One stu17 ratio . Also, cfication . Dual coHC cases . Howeverseconds) . Furtherseconds) . An interatories . On the ratories .HER2 gene status. The chromogenic in situ hybridization (CISH) assay using a DAB chromogen and H2O2 substrate system for horseradish peroxidase (HRP)-based signal detection has been evaluated and the value of this assay for assessing HER2 status has been demonstrated [HER2 gene signal. However, with the current CISH method, the assessment of the HER2/CEN 17 ratio is conducted by enumerating HER2 and CEN 17 separately using two different tissue sections.There are alternatives to FISH for determining nstrated ,17-22. Cin situ hybridization (ISH) methods use autometallography and enzyme metallography: 1) Nanogold® with gold enhancement in situ hybridization (GOLDFISH) [in situ hybridization (SISH) [HER2 gene signal. On the other hand, the SISH method produces discrete metallic silver black signals. Horseradish peroxidase (HRP) of the detection system reacts with silver acetate, hydroquinone, and H2O2 and deposits metallic silver particles at the reaction site. The reaction product can be seen as discrete black dots under a brightfield microscope. Advantages of SISH include the high sensitivity for detection of single gene copies, the high resolution for quantifying DNA targets, and the high contrast with tissue counterstaining for visual separation of the signal and tissue morphology [Other brightfield microscopy OLDFISH) ,24 and 2n (SISH) -27. The HER2 SISH assay was evaluated for assessing the inter-observer interpretative reproducibility of the HER2 gene status of 99 clinical cases when compared against the reference standard FISH results [HER2 FISH and single color HER2 SISH was 96.0%  and the discrepancies were mainly observed among tumors with the heterogeneity of tumor cell populations [HER2 and CEN 17 on the same tissue section like two-color FISH assays. Dual ISH staining for HER2 gene and CEN 17 would be beneficial for analyzing chromosome 17 aneusomy and for delineation of cases displaying genotypic intratumoral heterogeneity.Recently, an automated  results . Overallulations . AdvantaHER2 status reproducibly is the use of automation for conducting the test in the same manner among different laboratories located in different parts of the world. Thus, as a step toward standardizing HER2 testing, our objective was to develop an automated brightfield double in situ hybridization (BDISH) assay for simultaneous detection of HER2 and CEN 17 DNA targets on formalin-fixed, paraffin-embedded breast cancer tissue samples. Using this method, HER2 status testing can be conducted in a simplified manner for more accurately identifying the patients who are eligible for trastuzumab therapy and potentially leading to the improvement of breast cancer patient care in the future.One prerequisite for testing HER2 status and BT-474 is a breast ductal carcinoma with amplified HER2 status (50–60 copies of HER2) and chromosome 17 polysomy [® Plus glass slides .MCF7 and BT-474 xenograft tumors were utilized for optimizing the BDISH assay. MCF7 is a breast adenocarcinoma cell line with non-amplified polysomy . ParaffiHER2 status by FISH using the PathVysion® HER-2 DNA Probe Kit  at the Cleveland Clinic Foundation. However, it should be noted that non-consecutive tissue sections were used for FISH and BDISH analyses.Ninety-four (94) breast cancer cases were used from the Cleveland Clinic Foundation and the Cleveland Clinic Lerner College of Medicine, Cleveland, OH, USA under IRB approved protocol. Tissue samples were routinely processed for paraffin-embedding after fixing with an alcoholic formalin fixative. All breast cancer cases had been previously tested for ® XT automated slide processing system  was used for the optimization and performance evaluation of the BDISH assay for HER2 and CEN 17 DNA targets. A protocol was established so that the entire assay procedure consisting of baking, deparaffinization, pretreatment, hybridization, stringency wash, signal detection, and counterstaining was completed as a one-step fully automated assay. Paraffin tissue sections on glass slides were baked at 65°C for 20 minutes prior to the deparaffinization step with EZ Prep™ (Ventana) at 75°C for 16 minutes. Deparaffinized tissue sections were pretreated with a combination of heat treatment with Reaction Buffer  and ISH Protease 2 or ISH Protease 3 (Ventana) to unmask DNA targets. Pretreatment conditions were chosen for optimal signal to noise ratio and tissue morphology preservation for the xenograft control slides as well as clinical case tissue slides.The BenchMarkHER2 and CEN 17 signal detection were conducted for a complete BDISH assay , a dinitrophenyl (DNP)-labeled, nick-translated repeat deleted DNA probe was applied to the glass slide for co-denaturing the probe and target at 95°C. Then, the hybridization step was conducted at 52°C for 2 hours. After 3 stringency wash steps were performed at 72°C with 2× SCC (Ventana), tissue sections were incubated with monoclonal rabbit anti-DNP antibody (Ventana) for 20 minutes and then with HRP-conjugated anti-rabbit antibody for 16 minutes at 37°C. The metallic silver deposit for HER2 ISH signal was developed using silver acetate, hydroquinone, and H2O2 reaction in the presence of HRP using the ultraView™ SISH Detection Kit (Ventana). For CEN 17 detection, the INFORM Chromosome 17 Probe (Ventana), a DNP-labeled oligoprobe, was applied to the tissue sections, denatured at 95°C and hybridized at 44°C for 2 hours. Then, after 3 stringency wash steps at 59°C with 2× SSC, tissues were incubated with rabbit monoclonal anti-DNP antibody for 20 minutes and then with an alkaline phosphatase (AP)-conjugated anti-rabbit antibody for 12 minutes at 37°C. Finally, the signal for CEN 17 was visualized with a fast red and naphthol phosphate reaction using ultraView Red ISH Detection Kit. Diaminobenzidine (DAB) chromogen and H2O2 substrate reagents from the ultraView Universal DAB Detection Kit (Ventana), 5-bromo-4 chloro-3-indolyl phosphate (BCIP) substrate and nitro blue tetrazolium (NBT) oxidant reagents from the ISH iVIEW™ Blue Detection Kit (Ventana), and a ready-to-use tetramethyl benzidine (TMB) solution  were also evaluated for CEN 17 signal detection of the BDISH application. Because both HER2 and CEN 17 probes were labeled with the same DNP hapten, CEN 17 signal detection was completed without DNP-labeled CEN 17 probe after HER2 signal detection to ensure that the anti-DNP antibody of CEN 17 detection didn't recognize the DNP hapten of HER2 probe.Sequential ISH procedures for HER2 and/or CEN 17 targets were counterstained with Hematoxylin II (Ventana) for 4 or 8 minutes and Bluing Reagent (Ventana) for 4 minutes. Counterstained slides were first rinsed with distilled water containing DAWN®  for removing LCS from slides and then rinsed with distilled water until soap was removed completely from the slide. Slides were blotted very gently with paper towels and completely dried at 45°C or 65°C in the oven for at least 15 minutes. One drop of Cytoseal™ 60 (Richard-Allen Scientific) was applied onto a dried slide and a glass coverslip was carefully placed onto the slide. Excess mounting media was removed from the slides by gently pressing the slides against paper towels. Different coverslipping methods were also evaluated for preserving the fast red staining during the assay development. BDISH results were observed with a Nikon ECLIPSE 90i microscope  equipped with Nikon digital camera DXM1200F (Nikon) without oil immersion objective lenses, up to 60×. However, for presentation purposes, mainly comparing to FISH images taken with a 100× objective lens, brightfield photographs contained in this report were obtained using a 100× oil immersion objective lens.Single or double stained tissue sections for HER2 and CEN 17 targets of xenograft tumor controls as previously described [PathVysion HER-2 DNA Probe Kit was used for the FISH test for escribed . PhotogrHER2 amplification with the PathVysion assay (Negative: HER2/CEN 17 < 2.0 and Positive: HER2/CEN 17 ≥ 2.0) and using the ASCO/CAP guideline criteria  with or without the equivocal cases. Scoring BDISH slides was conducted by 4 observers , who were experienced with scoring HER2 FISH slides. Scoring occurred at different sites and at different occasions using different microscopes. Each individual observer evaluated the set of slides at their own pace and judgement. No scores were provided by the observers when the staining quality was deemed not adequate. There was no communication among observers regarding their scoring experience of the BDISH slides. Concordance data of FISH scores vs. consensus BDISH scores among 4 observers and FISH scores vs. individual BDISH scores by 4 observers were determined using SAS 9.1  in calculating frequency tables and Kappa statistics. The consensus among observers was defined as the agreement of three or more observers on a given observation. Scoring of BDISH assays was also analyzed for the sensitivity and specificity against FISH scores with the historical scoring method and the ASCO/CAP scoring method without the equivocal cases. Discordant cases were investigated by a non-observer (HN) for possible causes using BDISH slides.Performance of the BDISH assay was compared to FISH results as the reference standard using the historical criteria for HER2 single ISH, CEN 17 single ISH, and HER2 and CEN 17 BDISH results with formalin-fixed, paraffin-embedded xenograft tumor sections are presented in Figure HER2 signal were recognized as black discrete dots in the nuclei with MCF7 xenograft tumor  even though the same hapten was used for the sequential hybridization method. For image comparison of BDISH and FISH for HER2 gene and CEN 17, FISH images with MCF7 tumor and BT-474 tumor are presented in Figure HER2 genes are seen as red-orange dots and CEN 17 targets are seen as green dots.Images of r Figure  while amh Figure . Single r Figure  and BT-4r Figure . After ss Figure  and ampls Figure . Because® film coverslipper method  after air-drying slides (data not shown). The most common method of coverslipping tissue sections stained with fast red is the use of an aqueous mounting medium. However, this method did not produce crisp fast red staining for quantitative analyses of BDISH signals (data not shown). For the second color for the BDISH application, DAB, BCIP/NBT, and TMB detection systems that produce brown, blue, and green to blue final product, respectively, were evaluated. However, they did not provide sufficient contrast against the HER2 ISH black signal (data not shown).One successful way to preserve the fast red staining was the use of a toluene-based Cytoseal 60 mounting medium placed onto completely dried tissue sections prior to coverslipping with glass coverslips. We also confirmed that the red signal was successfully preserved with the Tissue-TekHER2 gene and CEN 17 with formalin-fixed, paraffin-embedded xenograft tumor sections, we applied the assay to 94 breast carcinoma cases and scoring BDISH slides was conducted by 4 observers . The consensus among observers was defined as the agreement of three or more observers on a given observation. With the historical scoring method (Negative: HER2/CEN 17 < 2 and Positive: HER2/CEN 17 ≥ 2.0)  (Table After optimizing the BDISH assay for 2) Table , the conHER2/CEN 17 ratios could be readily conducted. Non-amplified HER2 gene cases showed 0–4 copies of HER2 genes and 0–4 copies of CEN 17 depending on cell cycle stage and how each cell was cut within a tissue section  variegated different genotype tumor cell populations in the same area of tissue section  discordant cases of the BDISH slides presented at least some degree of the genotypic heterogeneity of tumor cell populations. In general, there were two types of the tumor cell heterogeneity with n Figure  and 2) sn Figure . Breast HER2 status testing is important for identifying breast cancer patients who may benefit from receiving trastuzumab therapy. Currently, in the United States, HER2 IHC methods are most commonly used for primary screening for HER2 status, and borderline cases are subjected to dual FISH for HER2 and CEN 17 to determine the HER2/CEN 17 ratio. Because the discordance rate between local and central/reference HER2 status testing with IHC and FISH is significantly high [HER2 status in breast cancer [HER2/neu testing guideline [HER2 status testing include: 1) automating the entire process for slide staining [HER2 testing process within experienced laboratories and pathologists that perform large numbers of HER2 tests [Accurate tly high ,31-33, ttly high . The Ameuideline . Two potstaining  and slidstaining -39 and 2R2 tests .HER2 status testing is to automate the assay procedure for HER2 IHC and HER2 FISH assays so that human errors can be diminished. HER2 IHC assays can be performed using an automated slide staining system, but HER2 FISH assays remain technically challenging and time consuming manual molecular diagnostic assays in most laboratories. An evaluator of FISH slides must have access to specialized fluorescence microscopy in a dark room. Because of unstable FISH staining characteristics, the signals of FISH slides can be bleached easily, even while reviewing and enumerating signals. Furthermore, digital images of the FISH slide need to be captured with a sensitive camera system for each patient case for the HER2 gene status record. Therefore, it is desirable to automate a tissue-based HER2 gene status test that can be observed with a regular brightfield microscope and that produces stained slides that can be archived.One way to improve the accuracy of HER2 and CEN 17 targets within the same nuclei of tissue sections with a manual dual brightfield ISH application [HER2 gene staining. However, based on published images [HER2 gene and CEN 17 presented in the current study are: 1) the automation of the ISH application; 2) the visualization of both HER2 gene and CEN 17 targets in the nuclei of the same cell; 3) the generation of discrete HER2 gene signals; 4) the ability to reproducibly detect endogenous HER2 and CEN 17 signals in the stromal tissues and lymphocytes as a reliable internal assay control; 5) the ability to visualise signal with brightfield microscopy with non-oil immersion lenses; and 6) the capability to permanently archive the slides.While the concept of multi-color brightfield ISH applications was published in 1990's ,41, it wlication . This dud images ,42, TMB HER2 and CEN 17 probes are co-hybridized for dual color HER2 FISH. However, for the BDISH assay, because the stringency conditions for the nick-translated HER2 probe and the CEN 17 oligoprobe were different, it was necessary to conduct sequential ISH staining steps for HER2 gene and CEN 17 targets. For CEN 17 ISH, we have optimized a new detection system with an alkaline phosphatase-conjugated antibody and fast red chromogen and naphthol phosphate substrate reaction. The fast red-based detection was selected to obtain a good contrast of CEN 17 ISH signal against the discrete black dots of HER2 SISH signal. DAB, BCIP/NBT, and TMB detection systems did not provide sufficient contrast against HER2 ISH black signal (data not shown). Because fast red precipitate is soluble in organic solvents, in general, aqueous mounting medium is used for coverslipping. However, the standard coverslipping method with aqueous mounting medium on wet tissue sections did not produce tissue sections with high resolution and therefore detailed tissue structure could not be observed (data not shown). A successful method to preserve fast red staining for CEN 17 and high resolution tissue morphology was, after completely dry the slides, to apply a toluene-based tissue mounting medium  for coverslipping with cover glass or to use a film coverslipper (Tissue-Tek® film coverslipper). Incomplete drying resulted in faint red background staining particularly around the fast red precipitate sites with this method. Interestingly, the use of aqueous mounting medium onto the dried tissue slides produced yellowish background staining on tissue sections and this method did not produce satisfactory results (data not shown).HER2 gene or CEN 17 and BDISH for both targets was evaluated with xenograft tumors. HER2 and CEN 17 copy numbers have been documented previously using the FISH assay [HER2 and chromosome 17 polysomy (3 copies of chromosome 17 per nucleus) while one of chromosome 17 with HER2 deletion (2 HER2 copies per nucleus) [HER2 amplification with 50–60 copies of HER2 genes and 4–6 copies of CEN 17 per nucleus [HER2 genes are located not only on chromosome 17, but also are translocated on other chromosomes [HER2 and CEN 17 copy numbers produced with the single target ISH and BDISH methods matched with previously reported results. As both probes are labeled with the same DNP hapten, our first concern was to determine if detecting specific signal for each probe was feasible. We confirmed that the fast red chromogen detection reagents did not produce red signal when the fast red ISH was performed without the CEN 17 probe after detection of HER2 by SISH (data not shown). Thus, the SISH detection and the fast red detection can be combined to perform a sequential double ISH assay with 2 probes labeled with the same hapten. Because the sequential BDISH application uses 2 specific stringency conditions based on the length and sequences of 2 probes, it is not necessary to design 2 probes that require the same stringency for co-hybridization, like double color FISH assays.The specificity of single ISH for SH assay . MCF-7 cnucleus) . BT-474  nucleus . Amplifiomosomes . HER2 anHER2 FISH scores and HER2 and CEN 17 BDISH scores by 4 observers were calculated for assessing the performance of the BDISH assay. There were 9 discordant cases  based on BDISH score disagreement with FISH scores, even by one observer. We have found that the number of equivocal cases influences the concordance rate with the ASCO/CAP scoring method. There were 4 equivocal cases based on FISH scores and all cases showed the BDISH score disagreement by at least 2 observers. A similar observation was reported with an international HER2 testing proficiency study [HER2/CEN 17 ratios between 1.7 and 2.3 that are close to the 'equivocal' defined by ASCO/CAP HER2 scoring method (1.8 – 2.2). They also stated "equivocal cases are difficult to interpret, even highly experienced and validated laboratories" [HER2 status assessment of breast carcinoma cases, heterogeneity of HER2 gene status was observed in 40 of 742 cases (5%) [HER2 testing results [HER2 status. Further clinical evaluations of HER2 and CEN 17 BDISH application with patient treatment outcome data are required for more accurate HER2 status assessment of breast cancer patients to be obtained.Concordance rates between a set of gold standard dual color cy study . In theiatories" . In one ses (5%) . It has  results . With cuHER2 gene and CEN 17 targets in formalin-fixed, paraffin-embedded tissue sections that is highly concordant to the FISH and is reproducibly interpreted among observers. Assessment of HER2 gene status can be conducted without the use of a specialized fluorescence microscope and the time required for completing HER2 gene status assessment can be shortened significantly. Furthermore, this application has the potential to be used for other gene targets, any combination of a gene and its chromosome centromere, and tissue section-based gene assessment tests including gene translocation studies. The use of BDISH technology allows the simultaneous analyses of two DNA targets within the context of tissue morphology observation.We have successfully developed an automated BDISH application for HN, BHW, ML, AEM, FG, MF, EW, TM are employed by Ventana Medical Systems, Inc. RRT and MD received grant support and honorarium for speaking from Ventana Medical Systems, Inc.LW, ML, JP, and RRT were responsible for identifying and prequalification of the clinical cases used in this study. HN and TMG were responsible for the BDISH assay development and feasibility studies, staining the clinical samples, and preparing the manuscript draft and image data. BHW and ML were responsible for the final assay development. AEM conducted all statistical analyses for the performance of BDISH assay. FG was the study coordinator and contributed intellectual content of the study. MF designed the probes. EW, MK, MD, RRT, and TMG critiqued the assay performance with their molecular histology expertise during the assay development. FPL, MK, MD, and RRT were the observers for scoring the clinical samples. All authors contributed intellectual inputs to the study. All authors read and approved the final manuscript.


Arabidopsis Polycomb repressive complex 2 (PRC2) subunits CURLY LEAF (CLF), EMBRYONIC FLOWER 2 (EMF2) and FERTILIZATION INDEPENDENT ENDOSPERM (FIE) repress the expression of FLOWERING LOCUS C (FLC), a central repressor of the floral transition in Arabidopsis and FLC relatives. In addition, CLF directly interacts with and mediates the deposition of repressive histone H3 lysine 27 trimethylation (H3K27me3) into FLC and FLC relatives, which suppresses active histone H3 lysine 4 trimethylation (H3K4me3) in these loci. Furthermore, we show that during vegetative development CLF and FIE strongly repress the expression of FLOWERING LOCUS T (FT), a key flowering-time integrator, and that CLF also directly interacts with and mediates the deposition of H3K27me3 into FT chromatin. Our results suggest that PRC2-like complexes containing CLF, EMF2 and FIE, directly interact with and deposit into FT, FLC and FLC relatives repressive trimethyl H3K27 leading to the suppression of active H3K4me3 in these loci, and thus repress the expression of these flowering genes. Given the central roles of FLC and FT in flowering-time regulation in Arabidopsis, these findings suggest that the CLF-containing PRC2-like complexes play a significant role in control of flowering in Arabidopsis.Polycomb group (PcG) proteins are evolutionarily conserved in animals and plants, and play critical roles in the regulation of developmental gene expression. Here we show that the  Arabidopsis thaliana, this transition is genetically controlled by several pathways, including the autonomous pathway, the photoperiod pathway and the vernalization pathway, which form a regulatory network The transition from a vegetative to a reproductive phase  is a major developmental switch in the plant life cycle that must be properly timed to ensure maximal reproductive success. In Arabidopsis is FLC, a MADS box transcription factor that quantitatively inhibits the floral transition FLC expression is delicately controlled by various activators and repressors. The autonomous pathway, which includes FVEFCAFLOWERING LOCUS D (FLD) FLC expression to promote flowering, whereas FRIGIDA (FRI) activates FLC expression to delay flowering FLC expression in response to a prolonged cold exposure  to accelerate flowering in ArabidopsisFLC, in the Arabidopsis genome there are five close FLC relatives including FLOWERING LOCUS M (FLM), MADS AFFECTING FLOWERING 2 (MAF2), MADS AFFECTING FLOWERING 3 (MAF3), MADS AFFECTING FLOWERING 4 (MAF4) and MADS AFFECTING FLOWERING 5 (MAF5); these FLC relatives also appear to repress the floral transition A key component in this regulatory network in FLC expression. Activation of FLC expression in the presence of FRI is associated with the H3K4 trimethylation and also requires deposition of the histone variant H2A.Z in FLC chromatin FLC expression partly through generating repressive histone modifications in FLC chromatin. FLD is involved in the H3K4 demethylation and deacetylation of FLC chromatin FCA functions closely with FLD and is involved in H3K4 demethylation in FLC chromatin FVE is partly involved in the histone deacetylation of FLC chromatin FLC chromatin by Type I and Type II arginine methyltransferases is also associated with FLC repression FLC repression FLC chromatin such as increased trimethylation of histone H3 at lysine 9 and H3K27, and H4R3 dimethylation Chromatin modification plays an important role in the regulation of FLC inhibits the floral transition partly by reducing expression of a key flowering-time integrator, FTFT was first identified as a component of the photoperiod pathway, which promotes flowering in response to increased day length FT expression is activated by CONSTANS (CO), another component in the photoperiod pathway FT is expressed in the vasculature FT locus and represses its expression, and thus antagonizes the activation by CO FT acts as a flowering-time integrator that integrates signals from the photoperiod pathway and the FLC-mediated flowering pathways to promote the Arabidopsis flowering. Recent studies indicate that chromatin modification may play a role in the regulation of FT expression. It has been shown that LIKE HETEROCHROMATIN PROTEIN 1 (LHP1) directly interacts with FT chromatin and represses FT expression Arabidopsis has revealed that this repressive mark is associated with FT chromatin FT chromatin and its role in FT regulation remain elusive.Drosophila. PRC2 is composed of four core proteins including Enhancer of zeste (E(z); an H3K27 methyltransferase), Extra sex comb (Esc), Suppressor of zeste 12 (Su(z)12) and p55, and deposits trimethyl H3K27 to silence the expression of homeotic genes in Drosophila , directly mediates the repression of AGAMOUS (AG) via H3K27 trimethylation and thus controls floral organogenesis CLF plays multiple roles in plant development, and also directly represses the expression of SHOOTMERISTEMLESS (STM) and a flowering gene, AGAMOUS LIKE 19 (AGL19), during vegetative development FLC repression VRN2 is required for FLC repression by vernalization treatment FLC expression in response to vernalization treatment EMF2, a relative of VRN2 and Su(z)12, also plays an important role in sporophyte development, and maintains vegetative development by repressing the floral induction EMF2-mediated floral repression are unclear Recent studies have also shown that CLF, an Arabidopsis PRC2-like complex subunits CLF, EMF2 and FIE repress the expression of FLC and FLC relatives including MAF4 and MAF5, and that CLF directly binds to and mediates the deposition of H3K27me3 in FLC, MAF4 and MAF5 chromatin. Furthermore, we show that during vegetative development CLF and FIE strongly repress FT expression, and that CLF also directly interacts with and mediates the deposition of H3K27me3 in FT chromatin. Theses results imply that PRC2-like complexes containing CLF, EMF2 and FIE deposit repressive H3K27me3 in and directly repress the expression of these flowering genes, and thus control the flowering program in Arabidopsis.Here we report that FLC repression FLC repression in Arabidopsis plants grown in normal conditions . In addition, the expression of FLC relatives such as FLM, MAF4 and MAF5, like FLC expression, is also regulated by chromatin modification FLC relatives. First, we addressed the role of CLF in the regulation of FLC and FLC relatives. Transcript levels of these genes were examined in seedlings of the clf-81 mutant carrying a lesion in the SET domain of CLF FLC, MAF4 and MAF5 were de-repressed in clf, whereas transcripts of FLM, MAF2 and MAF3 in clf remained at levels similar to wild-type Col , we first examined whether CLF directly interacts with the FLC, MAF4, and MAF5 loci. Specifically, genomic DNA was immunoprecipitated using an antibody recognizing GFP from seedlings of a 35S:GFP:CLF clf transgenic line in which GFP:CLF fully functions and its distribution mimics that of the endogenous CLF FLC-P2) around the transcription start site (TSS) and 5′ part of Intron I of FLC (FLC-I) were greatly enriched, whereas a 5′ promoter region 1.8 kb upstream from the TSS in FLC was not enriched , were not enriched , an H3K27 methyltransferase in the Esc-E(z) PRC2 complex AG and STMclf and wild-type Col seedlings, including H3K27 dimethylation, H3K27 trimethylation and H3K4 trimethylation. Levels of trimethyl H3K27 were strongly reduced in clf relative to Col  in Col and loss of CLF activities significantly reduced the levels of trimethyl H3K27, consistent with the derepression of FLC in clf  in which FLC expression is repressed FLC chromatin in these accessions in the absence of vernalization treatment FLC chromatin can simultaneously carry these two modifications as it is formally possible that these modifications could occur in two subpopulations of FLC chromatin and not in the same physical region of FLC. To examine whether FLC chromatin concomitantly carries both H3K4me3 and H3K27me3, we performed a sequential ChIP in which FLC chromatin from seedlings was immunoprecipitated first with anti-trimethyl H3K4 and second with anti-trimethyl H3K27. Both the region around TSS (FLC-P2) and 5′ part of Intron I of FLC (FLC-I) in part of the FLC chromatin concomitantly harbor H3K4me3 and H3K27me3 (FT-E) and the middle of FT (FT-I) in part of the FT chromatin simultaneously harbor H3K4me3 and H3K27me3 (Ta3ACTIN 2 (ACT2) carrying abundant H3K4me3 (data not shown) but lacking of H3K27me3  and the middle of genomic FT (FT-I) were increased upon loss of CLF activities  complexes containing EMF2 and FIE directly interact with and deposit into the FLC, MAF4, MAF5 and FT chromatin repressive trimethyl H3K27 leading to the suppression of active H3K4me3 in these loci, and thus repress the expression of these flowering genes. Given the central roles of FLC and FT in flowering-time regulation in Arabidopsis, these findings suggest that CLF-PRC2 complexes play a significant role in control of the Arabidopsis flowering.Our studies reveal that the FLC repression in Arabidopsis plants grown in normal conditions FLC repression because CLF directly binds to FLC chromatin and loss of CLF function leads to a reduction in H3K27me3 and FLC derepression. SWN, a CLF relative, may also play a role in FLC repression because low levels of trimethyl H3K27 in FLC chromatin have still been detected in clf seedlings , Esc, Su(z)12 and p55, and these components are evolutionarily conserved in animals and plants  The pression . TogetheFCA, FLD and FVE, and these genes do not form a linear pathway FLC expression  located at the bottom of Chromosome 5 CLF represses only MAF4 and MAF5, but not MAF2 or MAF3 in this gene cluster. The CLF-dependent H3K27me3 occurs in MAF4 and MAF5, but is absent from MAF3 and At5g65090 (the gene immediately downstream MAF5), suggesting that the H3K27 trimethylation in MAF4 and MAF5 is not the result of spreading from the neighboring genes. Furthermore, CLF specifically binds to MAF4 and MAF5, but not to MAF3 or At5g65090. This suggests that CLF is specifically recruited to the MAF4 and MAF5 loci, indicating that there are cis-regulatory DNA elements in these two genes that may function similarly to Polycomb-group response elements in DrosophilaFT expression during vegetative development, suggesting that a PRC2-like complex containing CLF, EMF2 and FIE represses FT expression. To date, all known PRC2 complexes in animals and plants contain four core components including p55 or a p55 homolog  has been shown to give rise to extremely early flowering independent of the photoperiods FT to be expressed at low levels for preventing precocious flowering and for the regulation of FT by the photoperiods. PRC2 subunits, likely functioning in the context of a CLF-PRC2 complex, maintain FT expression at basal lower levels in vegetative development, which may serve to provide some room for the elevated FT expression in response to photoperiods and thus enable the photoperiodic control of flowering time in plants.The PRC2-mediated transcriptional gene repressing mechanisms are conserved in animals and plants  as the endogenous control. Primers used are specified in Real-time quantitative PCR was performed on an ABI Prism 7900HT sequence detection system using SYBR Green PCR master mix (Applied Biosystems) as described previously Histone protein extraction and Western analysis were performed as described previously FLC, MAF4, MAF5 and FT were performed using SYBR Green PCR master mix (Applied Biosystems). Primers used to amplify FLC-P1, FLC-P2, ACTIN 2, TUB2 and TUB8 were described previously The ChIP experiments were performed as described previously The sequential ChIP experiments were performed as previously described Figure S1FVE represses MAF4 and MAF5 expression. Total RNAs were extracted from Col, fve and fca seedlings grown in long days. MAF4 and MAF5 were de-repressed in fve, but not in fca.(4.35 MB TIF)Click here for additional data file.Table S1(0.03 MB DOC)Click here for additional data file.


We postulated that cancer cells acquire immortality by activation of telomerase by the loss of such a gene. We have reported recently that a telomerase repressor gene may be located on 10p15.1 by deletion mapping using microcell-mediated chromosome transfer (MMCT), radiated microcell fusion (RMF), fluorescent in situ hybridization (FISH) and STS analysis. To independently confirm this result, we correlated expression of RNA component of telomerase (hTR) as a marker of telomerase expression by in situ hybridization with allelic loss in pulmonary carcinoid tumours. Unlike most malignant tumours, pulmonary carcinoids  are heterogeneous for telomerase expression. Loss of 5 closely spaced polymorphic markers on 10p15.1, especially D10S1728, were highly correlated with hTR expression. In an additional experiment, 10p15.1 showed higher and more significant correlation than any region of 3p where it has been predicted as another chromosomal location of telomerase repressor with allelic loss of the region. Our findings strongly suggest that 10p15.1 harbours a gene involved in repression of telomerase RNA component in human somatic cells and each putative repressor (on 3p and 10p) may act independently. © 2001 Cancer Research Campaign


The EORTC Lung Cancer Cooperative Group undertook a phase II study of paclitaxel in 25 chemotherapy-naive patients with malignant pleural mesothelioma. Paclitaxel was given intravenously at a dose of 200 mg m-2 as a 3 h infusion every 3 weeks, after standard premedication with corticosteroids and antihistamines. This regimen was well tolerated, with < 4% of cycles resulting in severe toxicity. No major objective responses were observed and ten patients had stable disease. Median survival time was 39 weeks and the 1 year survival rate was 30%. In conclusion, paclitaxel at the dose and schedule investigated in this trial had no major activity in the treatment of malignant pleural mesothelioma.


Heterozygous loss of relatively large chromosomal regions is a hallmark of the inactivation of tumour suppressor genes. Searching for deletions in cancer genomes therefore provides an attractive option to identify new tumour suppressor genes. Here, we have performed a genome-wide survey for regions exhibiting allelic loss in 24 commercially available breast cancer cell lines and four breast cancer xenografts, using microsatellite analysis. The assembled allelotype revealed an average fractional allelic loss of 0.34. A total of 19 arms had low allelic loss frequencies (<25%) and 17 arms had moderate allelic loss frequencies (25–50%). Five chromosomal arms were deleted in more than half of the breast cancer samples . Three of these frequently lost chromosomal arms had not been identified as such by comparative genome hybridisation, illustrating the higher sensitivity of microsatellite analysis for the detection of allelic losses. As we present allelic loss data of individual samples, our allelotype should not only aid the identification of new breast cancer genes but also provides a baseline for myriad studies involving these breast cancer cell lines. Genomic DNA was extracted using the Qiagen DNeasy kit. Microsatellite analyses indicated that all samples were unique and monoclonal. Of the 28 breast cancer samples, 22 (79%) were shown to be heterozygous for X-chromosome microsatellites , 9p21 (p16), 10q23 (PTEN), 13q12 (BRCA2), 16p13 (AXIN1), 16q22 (E-cadherin), 17p13 (p53), 17q21 (BRCA1), 18q21 (SMAD4), 19p13 (STK11), and 22q11 (hSNF5). Since constitutional non-neoplastic tissues were not available for any of the breast cancer samples, allelic losses were presumed based on statistical arguments. Multiple microsatellite markers were therefore designed within a 5-centiMorgan locus, thus reducing the odds of a deletion breakpoint between markers within a locus. Analysis of genomic DNA from 25 randomly selected non-neoplastic control samples revealed heterozygosity ratios for all microsatellite markers. Heterozygosity ratios generally were about 0.1 lower than those reported in the NCBI database, but higher ratios were also observed. The number of microsatellites that was analysed for each locus was such that the probability for a heterozygous sample to have a single allele size for each of the microsatellites within the locus was less than 5%, based on the heterozygosity ratios of the used markers. Allelic loss of a chromosomal locus was presumed when a breast cancer sample had a homozygous allele pattern for all microsatellites within the locus (with P<0.05 for each locus). In total, 146 microsatellites were analysed, resulting in an average of 3.6 markers per locus and an average P-value of 0.01. A homozygous allele pattern was observed for the heterozygous control samples at eight of 986 (1%) analysed loci, thus validating the statistical approach and implying an overall error rate for the complete allelotype of about 1%.We analysed 24 commercially available breast cancer cell lines and four breast cancer xenografts for allelic losses at all 41 non-acrocentric chromosomal arms, by PCR amplification of microsatellites. A single locus was analysed for each chromosomal arm, encompassing a known tumour suppressor gene at 5q21  and three (11%) of the 28 breast cancer samples, respectively, with one tumour being double mutant (Wasielewski and Schutte, manuscript in preparation, and (PTEN, BRCA2 and RB1, and BRCA1. Systematic mutational analyses of these tumour suppressor genes in this cohort of commercially available breast cancer samples should reveal whether these known genes indeed were the targets of the frequent allelic losses, or that other new tumour suppressor genes are likely to be located at these chromosomal sites.The wide range from 7% allelic loss at 1q to 82% at 17p that we observed in our breast cancer allelotype indicated a nonrandom deletion of genetic material, suggestive of the location of tumour suppressor genes at the sites of frequent allelic loss. Chromosomal arm 17p contains the


Hepatic artery ligation is used for the palliation of patients with malignant liver tumours. Collaterals aredeveloped rapidly and could to some extent explain why the growth is affected for only a short period.With intermittent dearterialization, collaterals seem to be avoided and possibly a more extended effectshould be expected. The most efficient period of dearterialization to avoid collaterals was studied in thisexperiment. Five groups of rats were treated with daily repeated transient dearterializations for 0 (n= 3), 60 (n = 6), 120 (n = 6), 180 (n = 6) and 240 minutes (n = 6) respectively for 5 days and comparedto another group (n = 3) that was permanently dearterialized. After treatment, celiac angiograms wereobtained. All hepatic arteries were reliably occluded and patent after 5 days of daily blockades in all buttwo rats. There were no collaterals demonstrable on the angiograms in the first four groups after 5 daysof intermittent obstruction of the arterial blood flow to the liver. After 240 minutes of dearterializationas well as after collaterals developed and were clearly demonstrated on the angiograms after six days.Liver enzymes were normal even after 4 hours of dearterialization. Repeated occlusions of the hepaticartery was reliably achieved with the implantable minioccluder. Repeated, transient dearterializationsfor 1, 2 or 3 hours could be performed without development of collaterals and without damage to theliver.


Screening for colorectal cancer (CRC) has shown to reduce cancer-related mortality, however, acceptance and compliance to current programmes are poor. Developing new, more acceptable non-invasive tests for the detection of cancerous and precancerous colorectal lesions would not only allow preselection of individuals for colonoscopy, but may also prevent cancer by removal of precancerous lesions.SEPT9, using a real-time quantitative PCR assay. To validate performance of SEPT9, plasma of 76 individuals  was assessed. Additionally, improvement of predictive capability considering SEPT9 and additionally ALX4 methylation was investigated within these patients.Plasma from 128 individuals (cohort I – exploratory study: 73 cases / 55 controls ) was used to test the performance of a single marker, In both cohorts combined, methylation of SEPT9 was observed in 9% of controls (3/33), 29% of patients with colorectal precancerous lesions (27/94) and 73% of colorectal cancer patients (24/33). The presence of both SEPT9 and ALX4 markers was analysed in cohort II and was observed in 5% of controls (1/22) and 37% of patients with polyps (18/49). Interestingly, also 3/5 (60%) patients with colorectal cancer were tested positive by the two marker panel in plasma.SEPT9 as a biomarker for colorectal cancer, they also show that methylated DNA from advanced precancerous colorectal lesions can be detected using a panel of two DNA methylation markers, ALX4 and SEPT9. If confirmed in larger studies these data indicate that screening for colorectal precancerous lesions with a blood-based test may be as feasible as screening for invasive cancer.While these data confirm the detection rate of  Colorectal cancer (CRC) is the second most frequent cancer in Europe and the US affecting 412,900 and 150,000 individuals in 2006, respectively A novel approach of molecular testing for CRC is the analysis of DNA in stool. In a study published by Imperiale et al. p16INK4, MGMT, GSTP1, CDH1, APC and TIMP3 among many others ALX4 and SEPT9 DNA in peripheral blood of colorectal cancer patients compared to controls SEPT9 methylated DNA, CRCs were predicted with a sensitivity of 72% and a specificity of 93% SEPT9 methylation in peripheral blood SEPT9 for the detection of early precancerous lesions we decided to further evaluate this marker in patients with polyps. Therefore, we designed this study, including an exploratory and a validation study, in order to further assess the performance of SEPT9 methylated DNA as a marker in the detection of colorectal precancerous lesions. In the validation study, we included also methylated ALX4 DNA to test if the predictive value for polyps increases if an additional marker is used, this has to be further validated, however. Since early non-invasive detection of polyps could be followed by endoscopic removal of precancerous lesions, this strategy might help to prevent more invasive colorectal cancers.Evidence is increasing that, apart from genetic alterations, epigenetic changes are of similar importance in the pathogenesis of CRC. Particularly tumor suppressor genes can be silenced by the methylation of CpG islands, which are found in the 5′ region of approximately half of all human genes The entire study population was assembled by two seperate cohorts. For cohort I (exploratory cohort), plasma was collected from 128 individuals with different clinical characteristics: healthy controls (n = 12), patients with symptomatic non-malignant bowel diseases with or without positive FOBT (n = 17), patients with a history of polypectomy or family history for colorectal neoplasia (n = 6), patients with chronic inflammatory diseases of the gastrointestinal tract (n = 20), 45 patients with colorectal precancerous lesions and 28 patients with CRC .For cohort II , plasma samples were collected from 76 individuals prior to any intervention: 49 patients  with colorectal precancerous lesions , from 22 healthy controls  without colorectal lesions and 5 patients  with CRCs  and the MagNAPure LC Total Nucleic Acids Large Volume Extraction Kit (Roche Diagnostics #03264793001) as previously reported 5′-GTAGTAGTTAGTTTAGTATTTATTTT-3′, the reverse primer was 5′-CCCACCAACCATCATAT-3′. Probe sequences were GTTCGAAATGATTTTATTTAGTTGC-FL and LC-Red640-CGTTGATCGCGG GGTTC-PH. The blocker sequence was 5′-CATCATATCAAACCCCACAA TCAACACACAAC-3′. A C3 spacer was introduced at the 3′ end of this sequence.SEPT9 methylation was determined using the HeavyMethyl technique 5′-CGTCGCAACGCGTACG-3′, sALX4r, 5′-CGCGGTTTCGATTTTAATGC-3′. Probe sequences were 5′-ACTCCGACTTAACCCGACGATCG-3′ and 5′-ACGAAATTCCTA ACGCAACCGCT-3′Genomic DNA was analysed by the MethyLight technique after bisulfite conversion as previously reported p-value of <0.05 was considered statistically significant The results of the MethyLight and HeavyMethyl assays were interpreted in a purely qualitative way as previously reported First, we determined the presence of a single DNA methylation marker in plasma from patients with colorectal precancerous lesions, CRC, in healthy controls without any colorectal pathology, patients with chronic inflammation, or other gastrointestinal diseases and patients with a high risk for CRC due to family history or positive FOBT . All metSEPT9 methylated DNA in 1 of 3 measurements was detected in 3 out of 12 healthy controls (25%), 28 out of 45 patients with polyps (62%), 25 out of 28 (89%) patients with colorectal cancers, 18 out of 20 (90%) patients with chronic inflammation and 6 of 6 high risk patients as well as 17 out of 17 patients with symptomatic diseases. Using the cut-off of at least 2 or 3 positive measurements only 2 of 12 healthy controls (17%), 9 of 17 symptomatic patients (53%), 4 of 6 risk patients (67%), 10 of 20 patients with inflammation (50%), 21 of 45 patients with colorectal precancerous lesions (47%) and 22 of 28 CRC patients (79%) were classified as positive  healthy controls. In contrast, SEPT9 methylation was significantly more frequent in patients with polyps (15 of 49 patients (31%); p = 0.01). Using the cut-off of at least 2 or 3 positive measurements with regard to the presence of SEPT9 methylated DNA in plasma, again only 1 of 22 (5%) healthy controls was classified as positive, whereas patients with polyps exhibited methylated SEPT9 DNA in at least 2 measurements in 6 of 49 (12%) cases. Cancer patients were positive in 2 of 5 cases using either classification  healthy controls and 27 out of 94 polyp patients (29%) as well as 24 out of 33 (73%) CRC patients were classified as positive. The difference in prevalence of SEPT9 DNA between polyp and control samples was statistically significant , 38 of the 49 polyp patients (78%) and in 4 of 5 (80%) cancer patients. Using the more stringent criteria (high specificity) the number of healthy controls with positive DNA detection decreased to 4 of 22 individuals (18%), whereas for patients with polyps 22 of 49 patients (45%) were considered positive (p = 0.02). Methylated DNA was observed in 2 of 5 cancer patients (In order to improve the detection rate of patients .SEPT9 + ALX4 and classifying a sample as “positive” if any of the single markers is “positive” (at least 2 of the 3 measurements), we observed 4 positive cases among the 22 healthy controls (18%), 25 positive cases among the 49 patients with polyps (51%) and 3 positive cases among the 5 cancer patients , whereas 18 of 49 patients with polyps (37%) and, again, 3 of 5 cancer patients were found to be positive. The difference between normals and polyps was highly statistically significant (p = 0.0013) .Next we turned to the analysis of marker performance with respect to the different types of polyps analysed in our study. Polyps were subclassified with respect to the clinically relevant size of <10 mm and ≥10 mm and histomorphological criteria . Patients with tubular or tubulo-villous adenomas irrespective of size were positive with regard to the presence of the marker panel in blood in 19 of 36 (53%) patients. The frequency of detectable methylated DNA increased in the subclass of patients with adenomas ≥10 mm . In the subgroup of polyps ≥10 mm and the presence of high grade intraepithelial neoplasia methylated DNA of the two markers was even observed in the plasma in 5 of 7 cases 71%; . OverallThe main problem of CRC screening today is low compliance in existing screening programmes. Despite the fact that CRC has all features which qualify this disease for mass screening, including high incidence and prevalence, high mortality in advanced disease, potential for cure in early stages, on average less than 30% of the eligible population in the US and Europe actually undergo these preventive procedures Based on the presence of certain genetic, epigenetic and related changes in the proteome of CRCs it has been tempting to assess the diagnostic accuracy of the presence of these changes in blood and stool for the purpose of CRC screening SEPT9 and ALX4 gene methylation as potential markers for CRCs ALX4 methylation in plasma was 83.3% and 70%, respectively SEPT9 methylation in plasma revealed a sensitivity of 70% and specificity of 90% for the detection of patients with CRC Another non-invasive approach is the detection of epigenetic changes in blood and/or stool of patients with CRC  and S4. ALX4 and SEPT9 methylation in CRC patients ALX4 and SEPT9 methylated DNA in plasma from patients with polyps versus healthy controls. In addition, the combined analysis of the two markers proved to be highly significant in the detection of colorectal polyps with a sensitivity and specificity reaching 71% and 95%, respectively, for the detection of advanced precancerous colorectal lesions. Thus, the performance of this marker panel was further enhanced after the detailed analysis of the histomorphological nature of the lesions that were observed in the patients with polyps. Accordingly, the combination of the two markers proved to be highly sensitive in the detection of advanced adenomas, which are defined clinically and pathologically as lesions that are ≥10 mm in size and exhibit features of potential malignant transformation .Similar to our previous reports on Recently, other groups have also analysed the presence of methylation markers in body fluids from patients with CRCs and adenomas, with a special emphasis on molecular stool analysis. However, the sensitivity of single methylation markers does not exceed 57% and in blood almost none of these markers were found so far  and S4. SEPT9 was increased among high risk patients (67%) and patients with chronic inflammation (50%). Thus, our exploratory study indicates that SEPT9 may be able to identify potentially curable early cancers or even precancerous colorectal lesions in asymptomatic individuals. However, patients with concomitant inflammatory diseases or other increased cancer risk should be directed to colonoscopy according to current clinical routine without non-invasive first line screening We also analysed the methylation status by including other symptomatic patients groups with non-malignant intestinal diseases. Methylation of In conclusion, our study presents the first evidence for a novel approach for the detection of advanced precancerous colorectal lesions using methylation markers in plasma. Advanced precancerous colorectal lesions are associated with an increased frequency of methylated DNA in the plasma. Based on this study, the diagnosis of colorectal precancerous lesions may be possible with a non-invasive, sensitive and reliable plasma-based test using a panel of methylation markers. If confirmed in a larger patient population, a diagnostic test based on this panel could improve early detection of cancerous lesions by allowing preselection of patients with precancerous colorectal lesions that could be subsequently be removed by colonoscopy, thereby offering the potential to prevent colorectal cancer.Figure S1Performance comparison of HM/Methylight assays on tissue samples. 198 Colorectal cancer tissues and 22 normal colon mucosa samples were analyzed by quantitative real-time PCR. Performance for the HM/Methylight is demonstrated by ROC plot analysis.(3.88 MB TIF)Click here for additional data file.Figure S2Performance comparison of MSP/Methylight assays on tissue samples. 198 Colorectal cancer tissues and 22 normal colon mucosa samples were analyzed by quantitative real-time PCR. Performance for the MSP/Methylight is demonstrated by ROC plot analysis.(3.83 MB TIF)Click here for additional data file.Table S1Classification table: Marker panel for detection of advanced polyps.(0.03 MB DOC)Click here for additional data file.Table S2Summary of statistical results.(0.04 MB DOC)Click here for additional data file.Table S3Performance of methylation markers in stool and blood for detection of colorectal adenoma.(0.04 MB DOC)Click here for additional data file.Table S4Results of multipanel assays with methylation markers for diagnosis of colorectal cancer in stool.(0.03 MB DOC)Click here for additional data file.


Rasgrf1 locus in mouse, a cis-acting sequence controls DNA methylation at a differentially methylated domain (DMD). While characterizing epigenetic marks over the DMD, we observed that DNA and H3K27 trimethylation are mutually exclusive, with DNA and H3K27 methylation limited to the paternal and maternal sequences, respectively. The mutual exclusion arises because one mark prevents placement of the other. We demonstrated this in five ways: using 5-azacytidine treatments and mutations at the endogenous locus that disrupt DNA methylation; using a transgenic model in which the maternal DMD inappropriately acquired DNA methylation; and by analyzing materials from cells and embryos lacking SUZ12 and YY1. SUZ12 is part of the PRC2 complex, which is needed for placing H3K27me3, and YY1 recruits PRC2 to sites of action. Results from each experimental system consistently demonstrated antagonism between H3K27me3 and DNA methylation. When DNA methylation was lost, H3K27me3 encroached into sites where it had not been before; inappropriate acquisition of DNA methylation excluded normal placement of H3K27me3, and loss of factors needed for H3K27 methylation enabled DNA methylation to appear where it had been excluded. These data reveal the previously unknown antagonism between H3K27 and DNA methylation and identify a means by which epigenetic states may change during disease and development.At the imprinted  Rasgrf1 locus in mice. It is required for methylation of nearby DNA sequences and can also influence the levels of local histone methylation. The methylation programmer has different effects on paternally and maternally derived chromosomes, directing DNA methylation on the paternal allele and histone H3 lysine 27 trimethylation on the maternal allele. These two methylation marks are not only mutually exclusive; they are also mutually antagonizing, whereby one blocks the placement of the other. Manipulations that cause aberrant changes in the levels of one of these marks had the opposite effect on the other mark. These observations identify novel mechanisms that specify epigenetic states in vivo and provide a framework for understanding how pathological epigenetic changes can arise, including those emerging at tumor suppressors during carcinogenesis.Methylation of DNA and histones exert profound and inherited effects on gene expression. These occur without changes to the underlying DNA sequence and are considered epigenetic effects. Disrupting epigenetic states can cause developmental abnormalities and cancer. Very little is known about how locations in the mammalian genome are chosen to receive these chemical modifications, or how their placement is regulated. We have identified a DNA sequence that acts as a methylation programmer at the  Within this region, there is a differentially methylated domain (DMD) that is subject to acquisition of epigenetic modifications, typically DNA methylation and histone modifications. These modifications are placed in a parent-of-origin specific manner and impose an epigenetic state that dictates allele-specific gene expression at imprinted loci In mammals, imprinted loci are expressed from only one allele. Accompanying and controlling monoallelic expression are allele-specific epigenetic modifications influenced by an Rasgrf1 locus. The ICR, located 30 kbp upstream of the transcriptional start site, is a binary switch consisting of a repeated element and the DMD. The repeated element functions as a methylation programmer, that is necessary for the establishment and maintenance of DNA methylation at the DMD on the paternal allele and sufficient for establishing gametic imprints in both germlines . However, in mice bearing a deletion of the repeats that constitute the Rasgrf1 DNA methylation programmer, only the DNA methylation at the originally defined DMD was lost. The DNA methylation on the additional 1.6 kbp was unaffected, indicating that the range of action of the Rasgrf1 DNA methylation programmer in the tandem repeats is limited to the 400 bp proximal DMD in the male germline  using mouse embryonic fibroblasts (MEFs) and antibodies specific to H3K9me2, H3K9me3, H3K27me2, and H3K27me3. Our initial tests were controls to verify that the antibodies detected histone modifications with proper specificity . For theWe then extended our H3K27me3 and H3K9me3 analysis to six segments . We were able to distinguish the transgenic ICR from the endogenous copy because the transgenic repeats were flanked with loxP sites and had the same structure as the WT-flox allele shown in Rasgrf1, then we predicted our MEFs with a methylated transgene should exclude H3K27me3, whereas our MEFs with an unmethylated transgene should allow its placement. This is also precisely what we observed  after amplification and will be digested, whereas unmethylated templates that underwent bisulfite conversion of either CG in the recognition site to TG will resist digestion. There should be an equal amount of digested and undigested PCR product when the maternal allele is completely unmethylated and the paternal allele is completely methylated. This is what we saw in embryoid bodies and blastocysts that were heterozygous respectively for the Suz12 and Yy1 mutations. This indicated that our COBRA assays accurately reported the presence of both methylated and unmethylated templates expected in these Suz12 and Yy1 expressing materials; however, it is not clear why Suz12 heterozygous ES cells did not show this pattern. When we performed COBRA analysis on SUZ12-deficient embryoid bodies (EB) that had differentiated for six (P6) or nine (P9) days in vitro , suggesting that at Rasgrf1, other CTCF binding partners and functions might be more important, such as YY1.We do not know how DNA and H3K27 methylation antagonize each other's placement; however, the literature highlights several molecular and developmental events, as well as protein factors that may be involved. Among these is the transcriptional state that is known to influence which of two mutually exclusive histone modifications is placed by the competing activities of polycomb (PcG) and trithorax (Trx) group proteins  Rasgrf1 , most liDM1 locus in humans, a repetitive element is associated with heterochromatin accumulation Rasgrf1 DMD and repeat sequences DM1 repeat also is a CTCF-binding insulator. CTCF appears to restrict the boundary of heterochromatinization at DM1, but it is not known if CTCF has a similar effect at Rasgrf1. Sequences with appreciable similarity to the Rasgrf1 tandem repeats are not abundant in the mouse genome. However, the Rasgrf1 repeat unit has striking similarity to the B repeat sequences on Xist . Previous reports describe the Suz12 mutation and preparation of homozygous ES cells and embryoid bodies Yy1 mutation and preparation of trophoblast outgrowths Mice used for DNA methylation analysis across the 12 kbp interval were F1 progeny of PWK and 129S4SvJae parents. Polymorphisms in these strains facilitated the assignment of a given clone from bisulfite PCR to one of the two parental alleles. Mice used to prepare MEFs for ChIP analysis across the 12 kbp interval were from strain 129S4SvJae backcrossed to C57BL/6 and included wild type animals, animals carrying a repeat deletion http://www.millipore.com for certificates of analysis for each catalog item and lot number). The DNA recovered after ChIP was used for Q-PCR with input chromatin and mock immunoprecipitations without antibody serving as controls. Q-PCR was performed in triplicate with SYBR green detection using primers listed in MEFs from 13.5 day old F1 embryos of C57BL/6 and 129S4Jae parents were used for ChIP analysis as described in BstUI digestions. In this assay, cytosine methylation enables digestion, whereas absence of methylation prevents it. Assays for DNA methylation using HhaI digested DNAs were described Treatment of genomic DNA with bisulfite was performed as previously described Figure S1CpG dinucleotides and methylated DNA centered at the DMD. A,B,C. The distribution of A, T, C and G over the 220 kb cluster show that there is a predominant accumulation of cytosines over the DMD and repeat region. The DMD repeat region has large amount of C and G together. There are two CpG islands in the region: one, which is the DMD and the other, which is in the promoter region of Rasgrf1 (bottom panel). D. Southern blot analysis of the two CpG islands using methylation sensitive restriction enzymes and tail DNA show that there is monoallelic methylation at the DMD (left panel) but no methylation of the promoter region CpG island (right panel). P (PstNI), N , E (EcoRI), Br . Bands diagnostic for the methylated (+) and unmethylated (−) states are indicated.(0.55 MB TIF)Click here for additional data file.Figure S2Specificity controls for antibodies used in ChIP. Representative gel analysis of ChIP results indicating the specificity of the antibodies for the histone modification analysis in this study. Antibodies specific to H3K9me2, H3K9me3, H3K27me2, and H3K27me3 show enrichment for H3K9me3 and H3K27me3 at the DMD. Positive control PCRs for H3K9me2 and H3K27me2 Charlie , H3K9me3(0.12 MB TIF)Click here for additional data file.Figure S3Rasgrf1 in wild type MEFs show that paternal DNA methylation (green) is largely even over the region, while maternal DNA methylation (red) is absent over the DMD but present upstream and downstream. Strikingly, H3K9me3 and H3K27me3 are perfectly confined to the DMD. (B) Modifications in the paternal allele in +/RepΔ mice. DNA methylation (black) is lost from the DMD and downstream, allowing encroachment of H3K27me3 into these regions (red).Mutual exclusion of H3K27 and DNA methylation. H3K27 and DNA methylation data from (0.15 MB TIF)Click here for additional data file.Figure S4Rasgrf1 ICR. (A) Xist sequences, including the A, B, C, D and E repeats (17 kb) and Rasgrf1 sequences including the DMD and repeats (5 kb) were aligned in a dot plot matrix. (B) Detail of the dot plot matrix in A that includes the Xist B element and the Rasgrf1 repeats.Dot plot of Xist and the (0.24 MB TIF)Click here for additional data file.Table S1Primers used for PCR amplification.(0.06 MB DOC)Click here for additional data file.Table S2Clones sequenced for analysis in (0.08 MB DOC)Click here for additional data file.Table S3http://www.broad.mit.edu/seq_platform/chip/ and experimentally verified CTCF site data were downloaded from http://insulatordb.utmem.edu/browse.php. After filtering the H3K27me3 ChIP data for sites with a read score of two or higher, the data sets were added as custom tracks on the UCSC Genome Browser and intersected in the intervals spanning 17,553 known genes and 53 imprinted gene regions. The intervals examined included the 100 kb 5′ of each gene (+100), sequences between the 5′ and 3′ ends of the genes (G), 100 kb 3′ of the genes (−100), and the entire stretch from 100 kb 5′ to 100 kb 3′ of each gene region (+100 to 100). The number of times H3K27me3 colocalized with CTCF in the indicated intervals is reported. The frequency of colocalization per kb was calculated for each interval examined, and the values for each of the known gene intervals were used to calculate an expected value for the corresponding imprinted gene intervals, given the total number of kbp in each of the imprinted gene intervals examined. The number of observed and expected colocalizations in the imprinted intervals was then used in Chi-square analysis.Enhanced colocalization of CTCF and H3K27me3 at imprinted loci. Whole genome H3K27me3 ChIP data for imprinted and known genes in MEF cells were downloaded from (0.06 MB DOC)Click here for additional data file.Text S1Supporting methods and references.(0.04 MB DOC)Click here for additional data file.


FHIT (fragile histidine triad) gene on chromosome 3p14.2 is a candidate tumour suppressor gene. To define the role of the FHIT gene in the development of ovarian cancer, we have examined 33 ovarian carcinomas, 2 borderline tumours and 10 benign adenomas for the presence of FHIT gene alterations. FHIT transcripts were analysed by RT-PCR and sequencing. Aberrant FHIT transcripts were observed in 5/33 carcinomas (15%) and in 1 of 2 borderline tumours. Loss of normal FHIT transcript was observed in 5/33 carcinomas (15%) but not in 2 borderline tumours or 10 benign adenomas. Allelic losses at D3S1300 and D3S4103, both located within intron 5 of FHIT were detected in 5/24 (21%) and 5/25 (20%) informative ovarian carcinomas, respectively. Expression of Fhit protein was analysed by immunohistochemistry in 44 carcinomas, 19 borderline tumours and 16 benign adenomas. Loss or significantly reduced expression of Fhit protein was observed in 6/44 (14%) ovarian carcinomas but not in any of 19 borderline tumours or 16 benign adenomas. The impaired Fhit protein expression was significantly correlated with the loss of normal FHIT transcription. Most notably, loss of normal FHIT transcript and impaired expression of Fhit protein occurred only in serous adenocarcinomas of grade 2 and 3 . The present data suggest that inactivation of the FHIT gene by loss of expression is one of the important molecular events associated with the genesis of ovarian carcinoma, especially of high-grade serous carcinoma. © 2001 Cancer Research Campaign http://www.bjcancer.comThe


PAX2 is a transcription factor with an important role in embryogenic development. However, PAX2 expression was frequently identified in neoplasia responsible for the growth and survival of cancer cells. Due to alternative splicing of exon 6, exon 10 and exon 12 four isoforms of PAX2 are described so far.The expression of an intron 9 containing PAX2 splice variant was analyzed in neoplastic B cell and solid tumor cell lines as well as in primary tumor samples by quantitative RT-PCR. PAX2 proteins were detected by Western Blot in a subset of cell lines.All 14 lymphoma cell lines expressed an undescribed PAX2 splice variant containing the entire intron 9 sequence and the exon 10 sequence. This splice variant could also be detected in 35 solid tumor cell lines, in leukemia and lymphoma as well as in colon carcinoma and melanoma patient samples and in blood samples of healthy donors. Expression of this new splice variant on protein level was verified by Western Blot analysis.We discovered a previously undescribed intron 9 and exon 10 containing splice variant of PAX2 in B-cell neoplasia and in solid tumors on mRNA and protein level. The PAX gene family was first described in Drosophila and later found to be conserved across species . PAX genAlthough PAX2 is primarily expressed during embryonic development and expression is normally repressed upon terminal differentiation, PAX gene expression was frequently identified in tumor cell lines, including lymphoma, breast, ovarian, lung, and colon cancer, as well as in primary tumor tissue samples  and was Apoptosis was induced in cell lines following RNA interference to silence PAX2 expression, suggesting that endogenous PAX2 gene expression is required for the growth and survival of cancer cells ,10,7. ThFour isoforms of PAX2 are described so far. They are products of alternative splicing of exon 6, exon 10 and exon 12: Exon 6 is present in the PAX2a transcript and absent in the PAX2b transcript . InsertiHere we characterized a previously undescribed intron 9 and exon10 containing splice variant of PAX2 in neoplastic B cell lines and solid tumor cell lines as well as in tumor tissue.14 lymphoma cell lines  and 35 solid tumor cell lines  All human cell lines were purchased from DSMZ  and CLS . Cells were maintained in RPMI 1640 containing 10–20% FCS, 2% penicillin/streptomycin and 2% glutamine .Fifteen primary low grade lymphoma, 9 myeloma, 11 acute lymphoblastic leukemia (ALL) samples and 7 AML samples were taken from patients that underwent routine diagnostics like venipuncture or bone marrow aspiration. Primary tumor single cell suspensions were prepared by ficoll hypaque separation. The lymphoma and leukemia samples contained more than 80% of tumor cells; therefore no further separation was done. For multiple myeloma, CD138 positive cells were isolated using Mini MACS technology . Tumor cells were resuspended in guanidium thiocyanate (GTC) buffer and stored at -80°C. 12 melanoma  and 12 colon carcinoma tissue samples were obtained from patients that underwent surgery for their tumors. Tissue samples were collected and dissected under stringent sterile conditions to prevent RNA contamination and immediately frozen in liquid nitrogen. There were no specific inclusion criteria with exception for the leukemia samples. Only PAX2 mRNA expressing AML and ALL samples were included. All patients had given informed consent for the analysis. Approval by the appropriate ethics committee has been obtained  and analyses have therefore been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki. Blood samples of healthy volunteers served as negative controls.Total RNA was isolated by RNeasy Mini Kit including RNase-Free DNase Set . Reverse transcription and quantitative Real Time RT-PCR  was done as described elsewhere . Primer 2 concentrations are listed in table PCR conditions and target-specific final MgClAnalysis of RT-PCR expression data was done with the LightCycler software (version 3). Sample concentrations were calculated using the plasmid standard curve resulting in marker concentrations. All samples were analysed in duplicate. The average value of both duplicates was used as a quantitative value. To correct for differences of cDNA amount on a per-sample basis, results were provided as ratio to housekeeping gene porphobilinogen deaminase (PBGD) expression. Statistical significance was tested using SPSS 15.0 software. For comparison of PAX2 intron 9 specific mRNA expression levels significance was estimated by the 2-sided Mann-Whitney U test for comparison of two different groups.Western blots were performed on equal amounts of protein obtained by lysis of cells using MPer Protein Extraction Reagent . The protein concentration was measured by BCA method using BCA Protein Reagent . 50 μg protein extract was loaded onto a 10% SDS-PAGE . Following electrophoreses, proteins were transferred to nitrocellulose membranes, and then blocked with 1%BSA in PBST  overnight at 4°C. Blots were then probed with rabbit anti-PAX2 primary antibody  at 1:1000 dilution. After washing with PBST the membranes were incubated with anti-rabbit IgG antibody conjugated to horseradish peroxidase (HRP) at 1:5000 dilution . Signal detection was visualized using ECL chemiluminescence reagent . As a control, blots were probed with mouse anti-β-actin primary antibody  and HRP-conjugated anti-rabbit secondary antibody.RT-PCR analysis of the PAX2 transcript in 14 lymphoma cell lines using a forward primer lying in exon 9 and a reverse primer lying in exon 10 (primer set PAX2_1) showed different PCR products on gel electrophoresis figure :All lymphoma cell lines showed a band of 339 bp of varying intensity. A band of the expected size of 185 bp was detected only in the cell lines KM-H2, EHEB, L540 and to a lesser extent in the cell line DG75. Sequencing analysis of the 339 bp PCR products revealed that this product results from the insertion of the entire intron 9 sequence. Thus, these cell lines expressed an undescribed PAX2 splice variant containing the entire intron 9 sequence and the exon 10 sequence figure  with a sTo analyze, whether the new splice variant is also expressed in solid tumors, a panel of solid tumor cell lines was tested by RT-PCR with the same primer set spanning the intron 9 (PAX2_1). Analysis of the product size by gel electrophoresis showed, that 7 of the 8 melanoma cell lines, 7 of the 9 colon carcinoma cell lines and 1 of the 7 renal carcinoma cell lines expressed the intron 9 and exon 10 containing splice variant. The other cell lines showed only a band of 185 bp. Additionally, 5 of 5 breast carcinoma cell lines, 3 of 3 lung carcinoma cell lines and 4 of 4 thyroid carcinoma cell lines expressed this splice variant.Next PAX2 positive leukemia patient samples were analyzed: In all 11 ALL samples and all 7 AML samples the new splice variant could be detected on gel electrophoresis. Subsequently, samples from patients with low grade lymphoma and multiple myeloma were analyzed. All 15 low grade lymphoma patient samples and 7 of the 9 multiple myeloma patient samples expressed the intron 9 containing splice variant. The remaining 2 multiple myeloma samples were negative for PAX2 mRNA . However, 22 of 24 blood samples from healthy donors surprisingly were also positive for the intron 9 and exon 10 containing splice variant.-4 (range 1.42 × 10-4 - 7.61 × 10-2). Additionally, in all solid tumor cell lines intron 9 specific mRNA was detected. The median expression was 1.49 × 10-3 (7.96 × 10-5 - 1.04). Moreover, the expression of the intron 9 positive PAX2 isoform was analyzed in 12 melanoma  and 12 colon carcinoma patient samples as well as in 9 AML and 5 ALL patients. All leukemia samples, 11 of the 12 colon carcinoma and 7 of the 12 melanoma samples were positive for expression of intron 9 specific mRNA. The median expression level in solid tumor samples was 1.17 × 10-1 (range 1.43 × 10-2 - 5.44) and in leukemia samples 3.07 × 10-4 (range 1.22 × 10-5 - 1.3 × 10-2)  figure . The exp-2 (range 6.33 × 10-4 - 1.63 × 10-1)  figure . The medTo verify the expression of this intron 9 positive splice variant on protein level, PAX2 protein expression was examined by Western Blot analysis in whole cell extracts of 3 colon carcinoma cell lines  and 4 lymphoma cell lines . Protein bands corresponding to known PAX2 isoforms  could be found in all cell lines. Additionally, a band of approximately 37 kDa figure  was idenHowever, in blood samples of healthy volunteers bands corresponding to the known splice variants of PAX2 and to a lesser extent to the new splice variant could be also detected. Expression of PAX2 in lymphoid cells was also observed by others .Therefore, regarding PAX2 targeted therapies like vaccination strategies caution is needed.We found a previously undescribed intron 9 and exon 10 splice variant of PAX2 on mRNA and protein level in B cell neoplasia and solid tumors as well as in peripheral blood of healthy patients. This splice variant has a distinct and a shorter C-terminus than the known exon 10 containing splice variant PAX2c due to the deletion of the last 89 amino acid residues. Alternative processing represents an important mechanism for the generation of various protein isoforms with different functions from one genetic locus . The funFinancial Disclosure: Ulrich Keilholz is holding a patent for the use of PAX2 for cancer immunotherapy. All other authors have declared there are no financial conflicts of interest in regards to this work.Grant Support: EU Integrated Project Cancer Immunology and Immunotherapy, project: WP 02.03 Transcription factors PAX2 and PAX8 as new tumor antigens.AB has made substantial contributions to conception and design, acquisition of data, analysis and interpretation of data and wrote the manuscript; AR: has made substantial contributions to conception and design, acquisition of data, analysis and interpretation of data. SS have been involved in acquisition of data and revising the manuscript critically for important intellectual content; ET has made substantial contributions to conception and design and was involved in revising the manuscript critically for important intellectual content, UK: has made substantial contributions to conception and design, as well as analysis and interpretation of data and wrote the manuscript.


Percutaneous access to the biliary tract was chosen when an endoscopic approach was not possible . Visualization of stones was achieved radiologically in 32 patients and by ultrasound in 24. The procedure was successful in 47 of 56 treated patients (83.9%). Clearance of the biliary tract was obtained in 25 cases (53%), whereas in 22 cases (47%) complete clearing of biliary tract was obtained only after endoscopic extraction of fragments (17 cases) or percutaneous (5 cases). The median number of shock waves in each session was 1725 (range 300–3166), which were applied during one (n=30), two (n=22) or three sessions (n=4). The only complications were 1case of symptomatic hyperamylasemia and 3 cases of macrohematuria. In conclusion, extracorporeal lithotripsy combined with endoscopic and/or percutaneous treatment is a real alternative to surgery for difficult stones.


Between 2003 and 2005, highly pathogenic avian influenza A (H5N1) viruses caused large scale outbreaks in poultry in the Ho Chi Minh City area in Vietnam. We studied the prevalence of antibodies against H5N1 in poultry workers and cullers who were active in the program in Ho Chi Minh City in 2004 and 2005.Single sera from 500 poultry workers and poultry cullers exposed to infected birds were tested for antibodies to avian influenza H5N1, using microneutralization assays and hemagglutination inhibition assay with horse blood. All sera tested negative using microneutralization tests. Three samples showed a 1∶80 titer in the hemagglutination inhibition assay.This study provides additional support for the low transmissibility of clade 1 H5N1 to humans, but limited transmission to highly exposed persons cannot be excluded given the presence of low antibody titers in some individuals. Since their re-emergence in late 2003, highly pathogenic avian influenza A (H5N1) viruses have spread across the globe, reaching endemic levels amongst poultry in several countries. The continuing occurrence of sporadic human H5N1 infections has ignited worldwide concern about an imminent influenza pandemic with potentially devastating consequences, especially if a pandemic H5N1 virus would keep its current virulence in humans. This threat persists despite the emergence of pandemic H1N1 in early 2009, either through direct adaption of H5N1 to efficient human transmission, or through reassortment with the novel H1N1 virus in swine or in humans.Based on reported cases, the mortality of human H5N1 infections still exceeds 60% with most patients dying of rapidly progressive respiratory failure. The occurrence of mildly symptomatic and asymptomatic human H5N1 infections has been suggested by seroepidemiological studies after the 1997 H5N1 outbreak in Hong Kong Vietnam has been one of the countries hit hardest by influenza H5N1 with 111 human infections reported since 2003. Beginning in early 2004, culling programs were initiated in Vietnam to contain spread of H5N1 across poultry farms. These programs identified infected poultry and provided in culling of all poultry on farms where infected poultry was found. We studied the prevalence of antibodies against H5N1 in poultry workers and cullers who were active in the program in Ho Chi Minh City in 2004 and 2005 when large scale poultry outbreaks were occurring in and around the city.The sub-department of Animal Health HCMC (AH) performed active surveillance in poultry farms (any size) in Ho Chi Minh City (HCMC), to identify and subsequently cull ducks and chickens with H5N1 infection, between October 2004 and June 2005. During this period, clade 1 H5N1 viruses were circulating in southern Vietnam Poultry farmers were identified by AH as having worked on a farm where poultry tested H5N1 positive during the 6 months prior to April 2005. Adults (>15 years old) living or working in affected sites at least 1 week prior to identification of H5N1 infected poultry, were included in the study. All cullers who were actively involved in culling during the period December 2004–June 2005, were identified by AH. All poultry workers on positive sites and all cullers were visited by staff from the Institute of Preventive Medicine from Ho Chi Minh City in June and July 2005. Witnessed oral informed consent was obtained from all participants. The study and the consent procedure were approved by the Hospital for Tropical Diseases Ethical and Scientific Committee and the University of Oxford Tropical Research Ethical Committee.A questionnaire was made in English and translated into Vietnamese. Participants were asked about demographic characteristics, exposure to poultry, the use of protective gear during exposure, and the occurrence of clinical symptoms during the period of study.Serum samples from poultry were collected on site and directly transferred to the laboratory of AH for analysis. Samples were tested within 48 hours of collection.A single 5 ml blood sample was obtained from all participants in June or July 2005, transported to the laboratory of the Oxford University Clinical Research Unit (OUCRU) in HCMC, and, after separation of serum, stored in aliquots at −20°C. Aliquots of serum were sent to Hong Kong (MP and WL) on dry ice for analysis. As the time between start of poultry culling and serum sample collection was more than 3 months for all participants, only single serum samples were collected and tested.www.oie.int/eng/normes/mmanual/a_00037.htm) using chicken red blood cells and H5N1 antigen derived from strain A\Ck\Scot\59 . Samples from dead poultry were subjected to real-time RT-PCR for detection of the H5 gene Serum samples from poultry were analyzed by HI as described in the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals of the World Organization of Animal Health  (clade 1) and A/Vietnam/30850/05 (clade 2.3.4) with suppression of viral cytopathic effect as the end point Human sera which tested positive at any dilution in at least one of the two tests, were retested by HI using horse red blood cells A total of 65 positive sites were identified. Of these, one site had dying chicken which were confirmed by RT-PCR to be infected with H5N1. Two additional sites had dying chicken which could not be tested for the presence of H5N1 as they had been destroyed. All other sites had ducks with positive HI tests. The number of poultry on a given site varied from 2 to 1600. The number of samples tested per site varied between 1 and 31 and the number of HI positive samples per site varied between 1 and 30.A total of 183 poultry workers were included in the study. 89 (50%) were male (sex was not recorded for 5 poultry workers) and the median age was 36 years . The majority of infected sites contained ducks . Almost 317 cullers were involved in culling during the period December 2004–June 2005. 195 (61.7%) were male (sex was not recorded for 1 culler) and the median age was 42 years (range 22–58 years). The median time between the last exposure and serum sample collection was difficult to assess as most cullers were involved in culling of infected poultry during extended periods of time . The preWe studied the prevalence of antibodies against H5N1 among 500 poultry workers and cullers in the HCMC area in 2005. All poultry workers and cullers had a history of exposure to poultry with evidence of symptomatic infection (dying chicken with positive RT-PCR) and/or asymptomatic infection (HI positive ducks) with H5N1. None of the poultry workers or cullers had antibody titres ≥80 in microneutralization tests, which is the recommended threshold for serological evidence of H5N1 virus infection, indicating that transmission of H5N1 was low. These results are in agreement with studies performed amongst poultry workers and persons exposed to infected poultry in the household in Nigeria, Thailand and Cambodia Interestingly, three cullers showed positive antibody responses in one of the neutralization assays, in two cases below-, and in a third case at the lower threshold of recommended cut-off values. These neutralizing antibody responses were confirmed in a different laboratory using a clinical isolate representative of the circulating virus at the time of the study. According to current guidelines, a second serological test, in this case HI using horse blood, was performed on all samples showing a titer in one of the neutralization assays. HI antibodies were observed in these three sera, albeit at titers one dilution below recommended cut-off values for confirmatory testing The median time between the last exposure and serum sample collection for cullers was difficult to assess as most cullers were involved in culling of infected poultry during extended periods of time . This prApproximately 3% of pre-vaccine sera of US participants in an H5N1 vaccine trial had H5N1 neutralizing antibody in the microneutralization test and in HI assay using horse red blood cells The exact exposures of poultry farmers and cullers are difficult to assess. Dying chicken in an area and time where H5N1 is highly prevalent, is highly indicative of infection in chicken, and therefore of exposure to those involved in farming and culling on farms where chicken were dying. However, the exposure is less clear for individuals who were exposed to infected ducks only. Whilst in the early stages of the current H5N1 outbreak infected ducks were reported dying, the virus adapted rapidly to become asymptomatic in ducks In conclusion, whilst our study provides additional support for the low transmissibility of highly pathogenic clade 1 H5N1 viruses to humans, limited transmission to highly exposed persons cannot be fully excluded given the presence of low antibody titers in some individuals.