Multiple sclerosis is a chronic inflammatory demyelinating disease of the central nervous system with an important genetic component and strongest association driven by the HLA genes. We performed a pooling-based genome-wide association study of 500,000 SNPs in order to find new loci associated with the disease. After applying several criteria, 320 SNPs were selected from the microarrays and individually genotyped in a first and independent Spanish Caucasian replication cohort. The 8 most significant SNPs validated in this cohort were also genotyped in a second US Caucasian replication cohort for confirmation. The most significant association was obtained for SNP rs3129934, which neighbors the HLA-DRB/DQA loci and validates our pooling-based strategy. The second strongest association signal was found for SNP rs1327328, which resides in an unannotated region of chromosome 13 but is in linkage disequilibrium with nearby functional elements that may play important roles in disease susceptibility. This region of chromosome 13 has not been previously identified in MS linkage genome screens and represents a novel risk locus for the disease. IL2RA) and the interleukin-7 receptor α (IL7RA), have been proposed as strong candidates for MS susceptibility in several studies IL2RA and IL7RA genes exert much weaker effects than the HLA-class II genes, associations of these two genes with the disease have been confirmed in several independent MS cohorts.Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system mediated by T-cell responses to myelin antigens Identification of these genes occurred through the use of a genome-wide association study of several hundred thousand single nucleotide polymorphisms (SNPs) in a broadly selected Caucasian population. Due to the need to multiple-test correct for the large number of SNPs assayed, the false negative rate for associations is quite high. An alternate approach is a two-stage design whereby an associated gene is found in a narrow population by a genome-wide screen and then evaluated in other populations. This approach is most effective when the associated allele is enriched in the initial population, such as due to a population bottleneck, but still present in the outbred population. However, the large cost associated with individually genotyping hundreds of individuals is often cost-prohibitive for the initial population. A more cost-effective alternative to individually genotyping thousands of samples on high-density SNP microarrays is the use of pooling-based GWA studies In our case, the pooling-based approach is particularly attractive since it allows us to test for associations across the Spanish population at relatively low cost as compared to individual genotyping, hypothesizing that additional genetic loci involved in conferring risk of MS could be identified through pooling-based GWA studies in this highly unique population.A total of 242 unrelated patients with relapse-onset MS visited at the outpatient clinic of the Unitat de Neuroimmunologia Clínica (UNIC) were included in the study. All subjects were of Spanish origin and satisfied Poser's criteria for clinically definite MS www.affymetrix.com).Prior to quantitation all DNA samples were checked for quality using 2% agarose gel electrophoresis, and obviously degraded samples were excluded from the pooling analysis. Individual genomic DNA concentration of each subject was determined in triplicate with the Quant-iT PicoGreen dsDNA Assay Kit  according to the manufacturer's instructions. These triplicate values were used to calculate a median concentration for each individual DNA. Individual DNA samples were then added to their respective pools in equivalent molar amounts. Once created, each pool was diluted to 50 ng/µl with sterile water in preparation for genotyping. Samples were genotyped  on 9 replicate Affymetrix 500 K EA arrays, following the Affymetrix protocols  values were calculated for each quartet. RAS values correspond to the ratio of the A probe to the sum of the A and B probes , and provide a quantitative index correlating to allele frequencies in pooled DNA. These values yield independent measures of different hybridization events and are consequently treated as individual data points.We used a silhouette statistic to rank all genotyped SNPs, since there are six paired-probes per SNP. This particular test-statistics has been heuristically shown to perform well at identifying SNPs with large allelic frequency differences, without merging data from fundamentally different probes even if for the same SNP. Consequentially, this test-statistic was utilized as it avoids introducing unnecessary variance by averaging probe intensity data from probes with different hybridization properties. This silhouette statistic intuitively represents the mean of the distance of a point to all other points in its class versus points in the other class. Silhouette scores range from 1, where significant separation between data points has been achieved and cluster assignment can be made with confidence, to −1, where differences in allelic frequencies are less reliable. The calculation for a silhouette score is shown in equation (1):Here, the overall silhouette score, S, is the average of all of the individual silhouette values, s(i), for each of the measurements and N refers to the number of replicate measures. Shown in Equation 1, for a calculation of an s(i) value, b(i) refers to the mean distance of a point to all points not within its class and a(i) refers to the distance within its class. In this study, we have two classes, MS cases and matched controls. SNPs were ranked based on silhouette score, whereby the SNP with the highest score was ranked 1 and the SNP with the lowest score was ranked 428,867 using Affymetrix's Mendel3 libraries for the Affymetrix 500 K arrays. With this ranking, it is assumed that SNPs approaching a rank of 1 will have larger differences in allelic frequency.We applied two criteria to select candidate SNPs for validation in a replication cohort of cases and controls. A first criterion was to select the top 100 high-scoring SNPs from the arrays obtained after ranking SNPs using a silhouette statistic, as described above. A second criterion was to identify clusters of two or more SNPs located in close proximity and scoring in the top 1%, and subsequently select from each cluster the SNPs having the highest scores.2. This statistical model was chosen as it is analogous to the type of allelic associations that would detectable by a pooling-based GWA study.A Spanish Caucasian replication cohort comprised of one hundred unrelated relapse-onset MS patients attended at the outpatient clinic of the UNIC and one hundred controls from the hospital transfusion centre was used for validation of the selected top scoring SNPs. None of these MS patients and controls was part of the original cohort used for DNA pooling. Demographic and clinical characteristics of MS patients and controls are summarized in DNA samples from this replication cohort were quantitated at the National Genotyping Center  using the PicoGreen assay. An Illumina GoldenGate assay was utilized to individually genotype SNPs selected from the Affymetrix 500 K platform.Seven SNPs were discarded because of significant departures from Hardy-Weinberg equilibrium. Two samples were also discarded because their X chromosome genotypes were inconsistent with their reported sex.www.snpator.com) Quality control processes and allelic and genotypic association tests were performed using the SNPator package . A description of the demographic and clinical characteristics of the US replication cohort is shown in th position). As expected, there has been evidence that some probes may poorly discriminate and some probes may preferentially bind one allele over another. The use of the silhouette statistics avoids adding unnecessary noise resulting from averaging fundamentally different measurements. The purpose of this paper is not to compare or contrast different methods, but rather to utilize a proven method to gain insight into the genetic basis of MS.A schematic flow chart summarizing the main steps of the study design and analysis is represented in From the ranked list of SNPs and after applying the two selection criteria mentioned in −5 with an OR of 3 (2 = 0.93). The extremely strong linkage disequilibrium between these two markers allows us to conclude that the association of rs3129934 results from the HLA-DRB1 locus and that the association observed with rs3129934 does not represent a second MHC association signal.Our GWA study design and the pooling-based strategy is essentially validated by the fact that the single most significant SNP neighbors the HLA-DRB/DQA loci. Specifically, rs3129934 is approximately 200 kb from the HLA-DRB1 locus  and has  OR of 3 . Previou−3 and an OR of 1.9 ; rs7821848, SNP located upstream of the SNTG1 gene ; rs10925318, intronic SNP in the RYR2 gene (ryanodine receptor 2); rs4902496, SNP located in the 3'untranslated region of the PLEKHH1 gene  member 1); rs7204129, intronic SNP in the CDH13 gene ).Finally, five additional SNPs that were found to be associated with the disease in the Spanish replication cohort are the following : rs71416−10. The second most significant SNP was rs1327328 on chromosome 13 with an allele based p-value of 0.0030, replicating the previously mentioned p-value of 1.8×10−3. For rs1327328, the association is driven by an excess of the T allele in both MS relapse-onset populations. The neighboring rs7326018 also showed significance at the 0.05 level, and as before showed some linkage disequilibrium with rs1327328 . Just as before, haplotype analysis revealed association of the H1 and H2 haplotypes with the disease  and TT homozygosity  for SNP rs1327328 were associated with relapse-onset MS. Individual association analysis obtained for the whole US replication cohort is also shown in SNP rs1327328 was genotyped in an additional US Caucasian population of 178 MS cases (mean age (standard deviation): 37.0 (9.9); % women: 81%; % relapsing-remitting MS: 100%) and 658 controls (mean age: 38.0 (11.9); % women: 80% female). ENSG00000184837, ENSG00000165618 and LOC121727) and microRNAs . Within or close to some of these functional elements, there are SNPs that have been typed by HapMap. Linkage disequilibrium analysis between SNPs rs1327328 and rs7326018 and the SNPs mapping in the functional elements is summarized in LOC121727 (similar to Peroxisome assembly protein 12 (Peroxin-12) (Peroxisome assembly factor 3) (PAF-3)).As mentioned above, SNPs rs1327328 and rs7326018 lie in a position of chromosome 13 devoid of any known functional elements. As shown in IL7RA and IL2RA, have been shown to contribute to the non-HLA-related susceptibility to MS Despite a large number of studies on susceptibility genes for MS, these efforts during the last two decades have been notably disappointing. Several whole genome screens and many linkage and association studies have only shown one genetic region with high consistency, the HLA-class II region on 6p21 and in Caucasian MS patients −4. This is an overly conservative number since some SNPs do show linkage disequilibrium between each other and, thus, the assumption of test independence central to the Bonferroni correction is not met. Still, rs3129934 survives this Bonferroni corrected significance level and, likewise, is known to be in strong linkage disequilibrium with markers in the HLA-DRB1 locus In the present study we used a recently developed and validated DNA pooling strategy and SNP array genotyping of 9 replicate pools as a first step. As the main advantage, this approach allows cutting costs and hence permitted us to perform the study on a novel population in order to screen a large number of SNPs for allelic association. Due to the large number of SNPs some correction is necessary in order to account for multiple testing. We did not genotype our original population, and all p-values are obtained from new cohorts that are completely non-redundant with the original pooled populations. Under the most conservative of scenarios, the p-values reported in −3. A second SNP, rs7326018, located approximately 30 kb from rs1327328 and in strong linkage disequilibrium with the latter was also associated with the disease in this replication cohort. The fact that both rs1327328 and rs7326018 show strong evidence for association suggests that genotyping error is not leading to a false association. SNP rs1327328 was validated in the US Caucasian population, with an allele-based p-value of 0.003, below the 0.006 Bonferroni-corrected significance threshold.SNP rs1327328 showed the second strongest association with MS in the Spanish replication cohort with a p-value of 1.8×10E2F1E2F1 as the best characterized member, plays a crucial role in the control of cell cycle SNPs rs1327328 and rs7326018 are located in the long arm of chromosome 13, at 13q31.3, in a region that has not been previously identified in MS linkage genome screens. In addition, rs1327328 and rs7326018 have not been found associated with the disease in other GWA studies. Thus, this region represents a novel risk locus for the disease. As mentioned before, this region of chromosome 13 is largely unannotated with no functional elements in close proximity. As to what could be the cause of the associations detected for these SNPs, a possibility is that they are acting as markers of some nearby elements. Interestingly, rs1327328 and rs7326018 are in high linkage disequilibrium with other SNPs mapping within or close to a cluster of microRNAs. MicroRNAs are a class of small regulatory RNAs that regulate gene expression LOC121727. This locus is a possible gene with homology to the peroxisomal biogenesis factor 12 (PEX12). Of note, mutations in human PEX12 result in Zellweger syndrome Linkage disequilibrium is also significant between SNPs rs1327328 and rs7326018 and markers mapping within or close to locus Finally, 5 of the 8 SNPs that were validated in the Spanish replication cohort were not associated with the disease in the US population. Regarding the ethnic background of the MS patients that were part of the replication cohorts, they are all Caucasian, however, one probably has to consider different ethnic admixtures in the US Caucasian and the Spanish Caucasian MS cohorts. Additionally, there are regional differences between MS prevalence and it is possible that susceptibility alleles for MS in a Spanish MS population may not be prevalent in a US Caucasian population. Consequentially, failure to see association of the other SNPs does not provide definitive evidence of a negative association, and these SNPs may represent regions of potential susceptibility to MS in the Spanish population. Furthermore, none of the 8 SNPs validated in the Spanish replication cohort by individual genotyping were found associated with the disease in a recent genomewide study conducted by the International Multiple Sclerosis Genetics Consortium In summary, by using a pooling-based approach and high density SNP arrays we confirmed the well-known association of the HLA class II genes with the disease, and identified a novel risk locus for MS on chromosome 13. Nevertheless, these findings are of preliminary nature and future studies will be needed to validate these results on chromosome 13 and to further implicate the functional elements of this region in disease susceptibility.Figure S1Flow chart summarizing the different steps undertaken in the analysis of the study data. A ranked list of SNPs was obtained from the analysis of the SNP arrays performed on pooled DNA from 242 Spanish MS cases and 242 Spanish controls . After applying two criteria (arrows), 320 SNPs were selected for individual genotyping in an independent Spanish cohort of 100 MS cases and 100 controls (Spanish replication cohort (1)). The SNPs showing greatest significance  were genotyped in a US cohort comprised of 275 MS cases and 275 controls (US replication cohort (2)). Two out of 8 SNPs (rs3129934 and rs1327328) were validated in the US cohort. A third SNP (rs7326018) showed marginal association in the US cohort. SNP rs3129934 is located in close proximity to the HLA-DRB1 locus. SNP rs1327328 was genoptyped in an additional US cohort of 178 MS cases and 658 controls and is located in chromosome 13, in a region that represents a novel risk locus for MS.(0.69 MB TIF)Click here for additional data file.Table S1Association analysis of the remaining 312 SNPs selected from the pooled SNP arrays and genotyped in the Spanish replication cohort.(0.46 MB DOC)Click here for additional data file.


The prognosis for patients with recurrent glioblastoma is still poor with a median survival between 3 and 6 months. Reports about the application of carmustine (BCNU), one of the standard chemotherapeutic drugs in the treatment of newly diagnosed glioblastoma, in the recurrent situation are rare.2 BCNU on days 1 on 3 intravenously at our department for efficacy, toxicity and prognostic factors. Progression free survival and overall survival were estimated by the Kaplan-Meier method. The influence of age, Karnofsky performance status (KPS), tumor burden, pretreatment with temozolomide (TMZ), type of surgery for initial diagnosis and number of previous relapses on outcome was analyzed in a proportional hazards regression model.We performed a retrospective analysis of 35 patients with recurrent or progressive glioblastoma treated with 80 mg/mThe median age of the group was 53 years, median KPS was 70. Median progression free survival was 11 weeks , median overall survival 22 weeks (95% CI: 18-27). The rate of adverse events, especially hematological toxicity, is relatively high, and in 3 patients treatment had to be terminated due to adverse events . No influence of age, KPS, tumor burden, pre-treatment with TMZ and number of previous relapses on outcome could be demonstrated, while gross total resection prior to recurrence showed a borderline statistically significant negative impact on PFS and OS. These data compare well with historical survival figures. However prospective randomized studies are needed to evaluate BCNU efficacy against newer drugs like bevacizumab or the intensified temozolomide regime (one week on/one week off).In summary, BCNU treatment appears to be a valuable therapeutic option for recurrent glioblastomas, where no other validated radio- and/or chemotherapy are available. This un protein . HoweverTherefore, this study was performed to obtain further data about the efficacy and toxicity of BCNU in recurrent glioblastoma that could serve as a benchmark for newer drugs, and to identify possible prognostic factors.We performed a retrospective review of the MS ACCESS database of our department for all patients with high-grade gliomas who received chemotherapy with BCNU between 12/2003 and 05/2008, and identified 93 patients. The patients' records as well as histological and radiological examinations were re-examined, and patients with a histologically proven glioblastoma, and radiological evidence of recurrent or progressive disease according to the Mac Donald criteria [2 BCNU on days 1 to 3. The treatment cycle was repeated every 8 weeks for a maximum of 6 cycles. Patients were evaluated weekly for hematologic, renal, and hepatic toxicity. Treatment was terminated in the presence of either a neuroradiological or clinical progression or inacceptable toxicity .The treatment schedule consisted of intravenous application of 80 mg/mPatients underwent routinely neuroradiological and clinical evaluation for tumor response every 8 weeks or earlier, when clinical deterioration occurred. Radiological response was assessed according to the Mac Donald criteria  we definThe largest cross-sectional tumor area at start of the BCNU therapy was defined as the tumor burden and was the result of the multiplication of the largest cross-sectional diameters.Study endpoints included evaluation of progression free and overall survival. Progression free survival was defined as the interval from start of BCNU treatment to radiological or clinical progression or death, whichever occurred first. Overall survival was defined as the time span from start of BCNU until death from any cause. Patients' general practitioners were contacted between May and September 2008 to obtain the most recent follow-up information.Progression free and overall survival were estimated by the Kaplan-Meier method, censoring observations at the time of last follow-up if the respective event was not observed. Median follow-up was derived from the estimated censoring distribution. Prognostic factors  were all fitted together in an exploratory fashion into a Cox regression model, both as continuous and dichotomised predictors. Two-sided 95% confidence intervals were used and statistical significance defined as p < 0.05 based on two-sided tests.Thirty-five patients were included into the study. 34 patients had a histologically proven GBM, in one patient diagnosis of GBM was confirmed in the combination of histopathological, neuroradiological and clinical findings. There were 21 (60%) men and 14 (40%) women with a median age of 53 years (range: 27 to 77 years). 26 patients (74%) were older than 45 years and median KPS at start of BCNU treatment was 70 (range: 40-90), 37% of the patients had a KPS below 70. 	Tumor involved one lobe in 22 patients , two or more lobes in 10 patients  and was multifocal in 3 patients. The operative procedure at time of first diagnosis of GBM was a gross total resection in 15, a partial resection in 4 and a stereotactic biopsy in 16 patients. 31 (89%) patients received postoperative radiotherapy to limited fields and 24 (69%) additionally an adjuvant and concomitant chemotherapy with temozolomide. In 30 patients BCNU therapy was initiated after the first relapse, in four patients after the second relapse  and in one patients a circumscribed tumor progress was irradiated with LINAC) and in one patient after the fourth relapse . Median time span from initial diagnosis to start of BCNU therapy was 38 weeks (range: 11-189 weeks). For further details see also table 62 cycles of BCNU were administered, ranging from 1 to 4 cycles, with a mean of 1.8 cycles per patient. Bone marrow toxicity, coagulopathies, pulmonal and infectious complications were evaluated according to the common toxicity criteria for adverse events V3.0. In summary 7 patients developed one or more adverse events during BCNU therapy. 4 patients developed thrombocytopenia grade 3 or 4, 5 patients developed leucopenia grade 3 or 4 and 1 patient developed an anemia grade 3. Two patients developed infection grade 3 or 4 and one patients a thromboembolic event grade 4 associated with pulmonary embolism. One patient experienced an interstitial lung fibrosis grade 3 after the administration of the third cycle BCNU. All patients with bone marrow toxicities grade III or IV have been pretreated with temozolomide, have been administered two or more cycles of BCNU and have had a stereotactic biopsy. In three patients BCNU treatment was terminated due to adverse events .In two patients a partial response and in 19 patients a stable disease was observed. We found no patients with a minimal or complete response. 11 patients showed a progressive disease after application of the first cycle. In three patients evaluation of radiological response was not possible.Of 35 patients, 29 progressed within the follow-up period, 5 were alive and progression-free, and one patient died 28 weeks after initiation of BCNU with no data on progression available . 30 patients died, 5 were alive at the end of follow-up. Median progression free survival was 11 weeks , with a 6 months progression free survival rate of 13%  on progression free survival, only gross total resection showed a borderline statistically significant association with a higher risk of relapse . The rationale for reoperation is to reduce intracranial pressure, improve neurological status of the patient and possibly improve efficacy of adjunctive therapy. Additionally it offers the possibility of BCNU-impregnated wafer insertion into the resection cavity and in combination with irinotecan median survival rates of 13.5 months are reported . OverallStereotactic radiosurgery (SRS) is a treatment option for small recurrent glioblastoma. Median survival of patients undergoing single-fraction SRS is around 10 months -15 and cFor these reasons chemotherapy is still the most common treatment option for recurrent glioblastoma multiforme . BesidesHowever, the role of BCNU - one of the few drugs with proven activity against GBM - in this situation is poorly defined. Brandes et al.  performeOur results compare well with the reported 6-months progression free survival and overall survival rates reported by Brandes  and WongIt has to be considered that our study population was very heterogeneous with a high variation of tumor burden and a high proportion of patients older than 45 years, who had already received various therapeutic modalities and were not only treated for the first but also for a second or fourth relapse of the disease. Therefore, we investigated if age, KPS, tumor burden, number of previous relapses or pre-treatment with TMZ had any impact of patient's PFS or overall survival.6-Benzylguanine, a compound that reacts with MGMT by covalent transfer of the benzyl group to the active site-cysteine and renders by this way the tumor cells 2- to 14-fold more sensitive to alkylating agents in vitro and in vivo settings [6-Benzylguanine with temozolomide or BCNU are currently under way [As TMZ and BCNU are counteracted via the same resistance mechanism, we hypothesized that pre-treatment with TMZ could have led to a resistance against alkylating agents reducing the efficacy of BCNU. We found no statistically significant impact of TMZ pre-treatment on PFS or overall survival, so that BCNU is still a therapeutic option for patients progressive under TMZ as the role of alkylating agents in the development of chemotherapeutic alkylating drug resistance remains still unclear . Howeversettings  and phasnder way .The tumor burden at the beginning of BCNU therapy had no influence on PFS or overall survival. This is in line with the level II recommendations for the surgical management of newly diagnosed glioblastomas, that recommend that only maximal cytoreductive surgery (>98% of tumor) correlates with a significant effect on survival -32. TherBrandes , who alsIn summary, we could not identify an influence of age, KPS, tumor burden, pre-treatment with TMZ or number of previous relapses on PFS or OS in our patient population. The data do suggest a negative impact of a gross total resection prior to recurrence in terms of PFS and OS, This might be due to a negative selection of patients with extensive tumor growth after initial complete resection who are not amenable for reoperation.Based on these findings we would recommend for patients with recurrent glioblastoma, who are not enrolled into clinical studies, to start with the intensified temozolomide scheme due to less toxic effects and superior progression free survival figures compared to BCNU. However, as we found no objective prognostic factors of BCNU efficacy it represents a valuable therapeutic option after a further progression when no other validated treatment modalities are available and the high rate of toxicity, especially after pre-treamtent with temozolomide has to be considered. Prospective and randomized studies are needed to compare the efficacy of nitroso-ureas against newer drugs like bevacizumab to prove their therapeutic superiority in the recidive situation.The authors declare that they have no competing interests.TR was responsible for the conception and design of the study; TR and TP performed the collection and assembly of data; EG, TR, TP, MP, MT, GN performed the statistical analysis and interpretation of data; TR, EG and GN wrote the manuscript; TR, EG, TP, MT, MP and GN revised the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/10/30/prepub


We carried out an analysis of the Genetic Analysis Workshop 15 simulated Problem 3 data. We restricted ourselves to the present/absent phenotype. Linkage analysis revealed a very strong signal on chromosome 6. Association analysis revealed additional susceptible loci located on chromosomes 11 and 18. The latter two signals were subsequently verified with linkage analysis – but only after 20 replicates were pooled. Analysis of linkage disequilibrium patterns, in concert with family-based association tests, led us to infer the presence of a second chromosome 6 locus located in the vicinity of single-nucleotide polymorphisms 160–162. These analyses were carried out without knowledge of the model used to generate the simulation. In the last few decades the genes responsible for hundreds of simple Mendelian phenotypes have been identified and their variants characterized. Progress on complex diseases, however, has been much slower. With the advent of new technologies capable of quickly genotyping millions of single-nucleotide polymorphisms (SNPs), the prospect of making substantial inroads in understanding complex phenotypes has improved. Nonetheless, to characterize the genetic architecture of complex phenotypes fully, a researcher will need to employ the full armamentarium of traditional methods, including linkage analysis, association analysis, and family-based transmission tests.s of 9.03. A total of 100 replicates were simulated. Each replicate consisted of 1500 nuclear families with a pair of offspring with RA and a random sample of 2000 unrelated individuals each drawn from the offspring generation of families containing no RA cases. Further details can be found in Miller et al. [We chose to investigate the simulated Problem 3 data set. Briefly, the simulation was designed to mimic the familial pattern of rheumatoid arthritis (RA), including the effect of the DR ideotypes at the MHC on chromosome 6, a lifetime prevalence of 1.07%, a 3:1 female:male affection ratio, and a λN = 9187) SNP map. The evidence for linkage was evaluated with MERLIN software [We performed linkage analysis on the affected sib-pair nuclear families. We restricted our attention for the preliminary analysis to the "sparse"  by selecting the most severely affected sib. If both sibs were equally affected, we selected the first sib. This case sample was compared to the N = 2000 controls supplied by the data providers. Ordinary chi-squares were computed for all SNPs  in the sparse map.We carried out an association analysis on the same sparse SNP map. From each of the first 50 replicates, we formed a group of unrelated cases  that interfaces with the TRANSMIT package . The sofp-value of 0.003). By contrast, the linkage signal on chromosome 6 attained a maximum LOD of 90.33 at SNP 152. Moreover, the LOD scores were positive over the entire length of the chromosome. Figure Linkage analysis of Replicate 1 revealed a very strong signal on chromosome 6 and little else. Only one other chromosome attained a LOD score > 1.0 , we decided to pool the first 20 replicates and repeat the analysis. Figure LD analysis suggests that SNP 153 on chromosome 6 and HLA-DR locus are very close to one another and the effects attributed to SNP 153 are likely due to its nearly complete LD with HLA-DR  the following counts were observed: 0, 114, 2525, 357, 0, 4 and 2, 293, 938, 2767, 0, 0, respectively).2 coefficients between SNP 153 and the suspected proximal and distal association signal regions. We first estimated the haplotypes from the genotypes ignoring the phase information given by the data providers and then we used the phase information. When estimated from the genotypes of cases in Replicate 1, we found no compelling evidence of LD between SNP 153 and SNP 160 or 162 . The phase-known haplotypes also give no evidence of LD. For the SNPs 138/139 vs. SNP 153 estimates, however, we find evidence of significant LD by both estimates (the phase-known chi-square is 31.4 and the phase-unknown maximum likelihood estimates yield a chi-square of 25.6).The linkage analysis of chromosome 6 in the first replicate produced a massive signal. We were, nonetheless, surprised that for a chromosome that is modeled to have a gender-averaged genetic length of 197.5 cM, the LOD scores were positive at every SNP position. The case/control association analysis gave a hint that other susceptibility loci may be located under the large LOD peak show in Figure Z-scores are significant – especially SNPs 152–155 – it is noteworthy that the next four most deviant SNPs  are the same SNPs that gave a strong association signal.To further scrutinize the pattern of significant associations on chromosome 6, we performed an analysis of SNPs 120–165 with FBAT to determine whether there is preferential transmission at any of the sites that showed association. Figure This long-range LD – over a simulated distance of >3.7 MB – was unexpected and suggests that the association and FBAT signals at SNPs 138/139 are a consequence of the LD rather than an independent risk locus. Accordingly, if there is a second risk locus on chromosome 6, a location in the vicinity of SNPs 160–162 would seem to be favored.R2 < 0.1. For chromosome 6 this involved the removal of 506 (75%) of the 674 SNPs. The resulting LOD distribution (not shown) is virtually identical to that produced with all 674 SNPs . This is not the case if parental genotypes are removed. For chromosome 6 using all 674 SNPs, but no parental genotypes, the linkage is still detected although the maximum LOD score is only 20.9 and is incorrectly positioned at SNP 121. The trimmed map  attains higher maximum LOD score (112.2) but is also incorrectly positioned at SNP 502.To evaluate the influence of LD on the linkage analysis, we selectively removed SNPs so that all pairs within one Mb had a It is instructive, and perhaps sobering, to realize that there are occasions in which linkage analysis is essentially powerless to detect a signal  that is readily detected by association analysis in a sample approximately one-tenth the size. Cox and Bell  providedThe presence of LD is known to inflate linkage statistics and the problem is exacerbated when parental genotypes are unavailable . Our anaExcept to determine whether trimming SNPs in LD could decrease type I error in the absence of parental genotypes, as noted above, all of the work reported here was undertaken without knowledge of the generating model.True to what is known about rheumatoid arthritis, the major genetic effect appears to be due to HLA-DR alleles on chromosome 6. Three additional signals were detected in our blind analysis; a second chromosome 6 signal in the vicinity of SNP 160–162, a signal on chromosome 11 in the vicinity of SNP 389, and a signal on chromosome 18 in the vicinity of SNP 269. These latter two signals were best detected with association analysis. To be detected by linkage analysis, a prohibitively large sample size of families would be required.The author(s) declare that they have no competing interests.


Functional annotation of protein sequences with low similarity to well characterized protein sequences is a major challenge of computational biology in the post genomic era. The cyclin protein family is once such important family of proteins which consists of sequences with low sequence similarity making discovery of novel cyclins and establishing orthologous relationships amongst the cyclins, a difficult task. The currently identified cyclin motifs and cyclin associated domains do not represent all of the identified and characterized cyclin sequences. We describe a Support Vector Machine (SVM) based classifier, CyclinPred, which can predict cyclin sequences with high efficiency. The SVM classifier was trained with features of selected cyclin and non cyclin protein sequences. The training features of the protein sequences include amino acid composition, dipeptide composition, secondary structure composition and PSI-BLAST generated Position Specific Scoring Matrix (PSSM) profiles. Results obtained from Leave-One-Out cross validation or jackknife test, self consistency and holdout tests prove that the SVM classifier trained with features of PSSM profile was more accurate than the classifiers based on either of the other features alone or hybrids of these features. A cyclin prediction server- CyclinPred has been setup based on SVM model trained with PSSM profiles. CyclinPred prediction results prove that the method may be used as a cyclin prediction tool, complementing conventional cyclin prediction methods. Herpesvirus saimiri and Kaposi's sarcoma-associated herpesvirus Cyclins were first identified in early 1980s in the eggs of marine invertebrates Caenorhabditis elegans, Drosophila melanogastor and Homo sapiensArabidopsis genome Cyclin family proteins are 30 to 65 kDa in size, sharing considerable sequence heterogeneity but share some common structural motifs known as ‘cyclin fold’. However, the conserved ‘cyclin fold’ is also found in general transcription factors (for example TFIIB) and tumor suppressor retinoblastoma protein SVM is a supervised learning algorithm, which has been found to be useful in recognition and discrimination of hidden patterns in complex datasets i) could represent the Amino Acid Compositions (AAC), Di-Peptide Compositions (DPC) of a protein sequence, Secondary Structure Composition (SSC) or any other training feature. AAC gives a fixed input pattern length of 20 whereas DPC gives a pattern of length 400 light package In context to the current study, SVMs learn the features specific to the cyclin sequences and generate support vectors decisive for possible classification of any given sequence as cyclin. For the cyclin classification problem, a feature vector  was also performed for the training variables to find out the variables important for distinguishing cyclins from non cyclin sequences.The optimized classifier was trained with different properties of a non redundant dataset consisting of 68 cyclin (positive dataset) and 72 non cyclin (negative dataset) sequences . Different properties of sequences like amino acid composition and dipeptide composition, and PSI-BLAST results were used for training and optimization of the classifiers. Optimization of different classifiers was performed using Leave-One-Out (LOO) cross validation (CV) technique or jackknife test, Self-consistency and Holdout tests. The biasness of selected classifiers was checked by calculating prediction efficiency for an independent dataset sequences not used in the training as well as annotated sequences of Three iterations of PSI-BLAST were carried out at E value 0.001. The performance of PSI-BLAST was evaluated using jackknife cross validation method , where each sequence in the training dataset was used as a BLAST query sequence and remaining sequences were used as BLAST database. Thus, in the process of jackknife test, each protein sequence is used as a test sequence, and for other rounds; the sequence is included in the training dataset. In PSI-BLAST output, it was observed that out of 68 cyclin proteins, no significant hits were obtained for 8 proteins, thereby resulting in overall accuracy of 88.2%. This implies that similarity-based search methods alone may not be the best choice for functional annotation of proteins. Therefore, we decided to explore methods based on other protein features for the prediction of cyclin proteins.AAC (Amino Acid Composition) analyses of cyclin and non-cyclin protein training sequences  reveal dWe performed training-testing cycles recursively to develop SVM models based on different features as well as combination of all the features (referred to as hybrids) . InitialWe found that for the SVM models based on AAC features, the linear and polynomial kernels yielded similar accuracies . Optimizing the parameters with RBF kernel yielded an accuracy of 83.57% which is comparable to that of the other two kernels . Hence, The optimization with linear and polynomial kernels for PSSM profile based classifier model resulted in ∼91% accuracy for both the kernels. However, SVM optimized with RBF kernel augmented the accuracy to 92.14% (for γ = 1 and C = 32.5), which was the best performance amongst the classifiers based on individual features. It had a sensitivity of 92.64% and specificity of 91.66% with MCC of 0.842. The comparison of results obtained with all the four features; clearly demonstrate that the best classification efficiency was achieved by the PSSM based model which incorporates the information about residue composition, position specific substitutions and evolutionarily conserved residues in the protein sequences. Also, the efficiency of RBF kernel based discrimination between cyclins and non-cyclins as compared to other kernels for AAC, DPC and PSSM and ROC (Receiver Operating Characteristic) plot study  suggesteIn our efforts to further improve the prediction accuracy, we developed and evaluated eight hybrid SVM classifier models (hybrid-1 to hybrid-8), results of which are summarized in the The first hybrid-SVM model (hybrid-1) was developed using AAC and DPC features of the training dataset sequences. The prediction accuracy of hybrid-1 model was 81.42% with sensitivity of 82.35% and specificity of 80.55%, which was not better than that of standalone AAC and DPC based SVM models. The hybrids-2 and 3 were based on secondary structure predictions, amino acid and dipeptide compositions. We found that hybrid-2 has greater accuracy as compared to that of hybrid-3 ; also better than the accuracy of individual SSC or AAC based SVM models however, comparable to the individual DPC based model. Further, when the training was done using features from both AAC and PSSM (hybrid-4) or DPC and PSSM (hybrid-5), the accuracy increased to 91.42%, as compared to hybrids-1 to 3.However, amongst the hybrid-classifier models based on two features (hybrid:1–6), the best accuracy of 92.14% was achieved by combining the SSC and PSSM pattern information in hybrid-6 classifier, with a sensitivity of 92.64% and specificity of 91.66%. This SVM model has a MCC value of 0.842, highest amongst the hybrid-SVM classifier models.Considering the importance of conserved secondary structures in cyclins, we decided to use SSC in conjunction with the training features of the best hybrid-classifier developed till this stage other than hybrid-6 (i.e. hybrid-5 based on PSSM and DPC features). Hence, we developed another hybrid model using the information from PSSM, DPC and SSC features (hybrid-7), with a pattern length of 860 features. Unexpectedly, the accuracy of this hybrid model was 90.01% (γ = 2 and C = 10), lower than the accuracies of hybrid-5 (PSSM+DPC = 91.42%) and hybrid-6 (PSSM+SSC = 92.14%) classifier models. Another hybrid model of PSSM, DPC and AAC (hybrid-8) was developed with which we were able to achieve an accuracy of 91.42%, similar to that of hybrid-4 and 5. Thus, the accuracy achieved by hybrid-6 and PSSM based model were the best amongst all the hybrids and standalone models, further supported by the highest MCC of these models.We carried out self-consistency tests of the best models namely: PSSM, hybrid-6 and hybrid-8 to evaluate their learning capability. We used two different modes to carry out the self-consistency test  were assessed by holdout tests. The holdout test results are highly convincing in terms of accuracies and MCCs (∼0.8 for PSSM model and ∼0.7 for hybrid-6). The tests results have ruled out any skewness, biasness or variance in the results due to random splitting of the dataset. Also, as holdout tests simulate the random/blind test performance for a large dataset, the results obtained here reflect a strong discriminative power of the classifier.Upon comparing the prediction reliability with that of random prediction , it was For each SVM model, threshold-independent performance was measured by plotting ROC curve between TP rate (sensitivity) and FP rate (1-specificity) values. ROC indicates the performance of all SVM models optimized with best parameters. A. thaliana genome that were predicted earlier by a HMM based method developed by Vandepoele et al. A. thaliana genome, 8073 protein sequences, are yet poorly annotated and mostly described as ‘unknowns’. However, an elaborative analysis and supporting evidences may be needed from case to case before any significant remarks can be made regarding these predicted cyclins.To evaluate the unbiased performance of the PSSM based and hybrid-6 based SVM classifiers, an independent / blind-test evaluation was carried out on the test set of 54 PROSITE false-negatives  describing amino acid frequencies  revealedThese findings are in agreement with the observed prevalence of amino acids in cyclins and non-cyclins  which inComponent loadings for the first three principal components explain only 21% of the variability of the dataset . The higThe principal component loadings of PSSM profile reveal that PC1 is capable of gathering 27.7% of total variance of the dataset . In totahttp://bioinfo.icgeb.res.in/cyclinpred” The home page of the webserver is simple and designed using HTML and PHP . Overall, PSSM based model was able to achieve an accuracy of 98.5% as compared to 95% by hybrid-6.Thus, these results not only confirmed that optimized SVM is able to learn crucial features responsible for the accurate classification but also, it helps us to understand that prediction accuracy can be increased by providing more comprehensive information of a protein sequence. Moreover, our studies reiterate that biological patterns generally follow non-linear equations/functions which may not be easily predictable by conventional computational methods. This is fact is well exemplified by the classification task undertaken in our study in which we found that the models optimized with linear kernel functions are less accurate as compared to those trained with non-linear functions. For example, with linear kernel, the accuracy achieved in case of DPC based model was 81.43% as against 85.13% with RBF kernel. Similarly, PSSM based model achieved 92.14% accuracy when optimized with RBF kernel as compared to ∼91% with linear kernel.The reason behind better performance of the model developed by us is mainly because we have used a statistical learning method which generates prediction models by learning from large complex datasets, also taking care of over-fitting problem. In most of the cases, a small fraction of total examples in the training dataset were used to gather all the information required for generating the classification model, thus the original dataset is filtered to make more informative and representative dataset in terms of Support Vectors (SVs) of representative sequences. In the study, the number of SVs finally chosen by SVM for training (PSSM and hybrid-6 based models) are 53 and 77 (out of 140 training sequences) which is just 38% and 55% of the total dataset, respectively. In the hybrid-6 model, training with information from both – the PSSM and secondary structure information as input to the classifier enhances both the prediction accuracy and reliability of the models generated.Furthermore, the PCA analysis of the features has proven that the features which encapsulate comprehensive information about sequences help better segregation of training examples and enhances their correlation with the features under study. This is exemplified by the case of PSSM based model as compared to other feature based models.The advantage with the present prediction method lies in the fact that classifier is independent of any sequence or structural similarity to any known proteins. The absolute inheritance of cyclin domains or motifs in proteins is not a mandatory object for prediction. The method can even predict those cyclins which have high sequence variability like cyclin M, T or some G2-specific cyclins. The results obtained from current implementation of features are highly encouraging. In future, more cyclin specific features may be included for enhancing the efficiency of the SVM model and subfamily specific classifiers may be developed. To develop subfamily-specific SVM classifiers, sufficient number of sequences representing each subfamily will be required. Currently, the number of cyclin sequences from each subfamily, is too low for developing subfamily-specific classifiers. Almost all the subfamilies have less than or equal to five sequences per group in the training dataset. Only cyclin B subfamily is represented by more than 30 sequences . The lowest score is 0.8% identity with a Cytochrome b protein from Dictyostelium discoideum.Subsequently, the redundancy in the dataset was brought down to 30 percent using PISCES algorithm It has been reported that the performance of N-fold CV is not completely unbiased We performed pfscan search to look for PROSITE cyclin motifs in the 68 positive dataset sequences used in the SVM training. The cyclin motifs were found in only 35 out of the 68 cyclin sequences. We also found that 61 protein sequences had one of the cyclin associated Pfam domains. Comparisons of these annotated cyclin sequences showed that 6 proteins neither contain the Pfam cyclin domains nor the PROSITE cyclin motifs. This emphasizes the fact that all the known annotated cyclins are not represented by Pfam domain families and/or PROSITE signatures.light, an implementation of Support Vector Machines (SVMs) in C language was used for the current study. The SVMlight (http://svmlight.joachims.org/) allows optimization of SVM models by changing a number of parameters, including types of kernels  to perform the classification task. It is similar to solving a quadratic optimization problem (QP) and the decision function can be solved asi is the corresponding target value, which is +1 for cyclins and −1 for non-cyclins. αi is the value provided by QP. The separating hyperplane generated by SVM model is given by2 and satisfies the following conditions:SVMThe entire dataset of ‘N’ number of sequences was divided into training and testing sets for which SVM parameters were optimized by Cross Validation (CV) method. The parameters were optimized by CV over the training set by maximizing the accuracy and minimizing the training error. In the study, we implemented three methods of evaluation to check the performance of classifier models, namely- Leave-One-Out CV (LOO CV) or Jackknife CV test, self consistency and holdout tests.In the first method, the jackknife test is a CV technique in which one dataset sequence is used as a testing data  while the remaining dataset sequences are used as the training data to develop a model. This is iterated N times till each sequence in the dataset become the testing data exactly once. In each of the iterations, the parameters corresponding to the best accuracy are recorded and then averaged for the final overall evaluation of the model. We also performed self-consistency test in which the entire dataset is used for the training as well as for testing to validate the generated model. This test indicates the confidence level of the model as it inherits the internal knowledge of the training dataset. Self-consistency test was carried out in two modes, differing in the use of kernel parameters. In mode-1, the performances of the models were checked by using the best kernel parameters as evaluated by jackknife CV test performed earlier. In mode-2, the performances of the models were checked by using new kernel parameters to achieve the best accuracy during the self-consistency tests.The rationale behind using two modes was that mode-1 would provide the self-learning capability of the model on those parameters which have been obtained from cross-validation with jackknife test as well as this model will be used for prediction purpose whereas mode-2 would provide the self-learning capability of the model on those parameters which have not been involved any cross-validation or partitioning of the dataset and therefore, reflects the consistency of the prediction model.In the third method of evaluation, namely the holdout test, the dataset is randomly split into two equal halves, with approximately equal numbers of positive and negative dataset sequences. SVM is then trained with one of these subsets with Jackknife CV test to find best parameters. The performance is then evaluated by testing the optimized parameters on the second subset. This test indicates the prediction efficiency of the models for sequences independent of the training.We also evaluated the performance of the SVM model by the following measures:-Sensitivity: percentage of cyclin protein sequences that are correctly predicted as cyclins.Specificity: percentage of non-cyclin protein sequences that are correctly predicted as non-cyclin sequences.Accuracy: percentage of correct predictions, for cyclins as well as non-cyclin sequences.Mathew's Correlation Constant (MCC): employed for the optimization of parameters and evaluation of performance MCC = 1 signifies perfect prediction while MCC = 0 suggests completely random prediction. The above mentioned evaluations may be represented mathematically as given below.All the above mentioned measures are threshold-dependent i.e. the prediction performance is evaluated for a fixed SVM cutoff score or threshold. To calculate threshold-independent performance, a Receiver Operating Characteristic (ROC) curve was plotted between TP rate (sensitivity) and FP rate (1-specificity) Another important consideration is whether the present prediction method is better than a random prediction. To check the reliability of the method, we first calculated R, an anticipated number of proteins that are correctly classified by random prediction Subsequently, we also calculated S, the normalized percentage of correctly predicted samples better than random i.e. the method is independent of the scale of total samples in the dataset and R:Among all common statistical measures like accuracy, specificity and sensitivity, F1 statistic is a more robust measure as other measures can overstate the performance of the classifier. F1 statistic is a harmonic mean of precision and recall , given by equation 8:i is any of the 20 amino acids.The following equation was used to calculate AAC,j can be any of the 400 dipeptides.The fraction of each dipeptide in the protein was calculated by the following formulae,k can be any of the 3 secondary structures (helix/sheet/coil) and j can be any of the 20 amino acids.Secondary structure is an important feature of cyclins due to its characteristic helical domains assuming helix rich cyclin-folds, containing the cyclin-box This model was designed using PSI-BLAST v2.2.4 output. For each amino acid, PSI-BLAST give 20 substitution scores in the PSSM which provides the evolutionary information about the protein at the level of residue types. Three iterations of PSI-BLAST were carried out at cut-off E-value of 0.001. Each value in the PSSM represents the likelihood of a particular residue substitution at a specific position of a protein class and it was normalized between 0 and 1 using the logistic function as shown in equation 12 g(x) is the normalized value of x. Following this, the normalized matrix is organized into a composition matrix of fixed length pattern of 400 . Normalization was done for other features also.Where x is the raw value in PSSM profile and PCA leads to a linear combination (projection) of the original variables of high-dimensional data and thereby, is used to calculate orthogonal variables from raw data matrices. PCA analysis was performed to identify different feature element variables from AAC, DPC and PSSM that are important for distinguishing cyclins from non-cyclins. Thus, it was performed with the objective of obtaining new variables that are uncorrelated among themselves  from the original ones and to reduce the dimension of data to unity with the minimum loss of information. Using “princomp” function of MATLAB, we extracted the PCs by using the same data matrices which were also used as input for SVM training. The scatter plots of scores of first two principal components for AAC, DPC and PSSM matrices are shown in the Table S1Training data set sequences from different cyclin subfamilies used in training the SVMs(0.04 MB DOC)Click here for additional data file.Table S2List of organisms and corresponding cyclin sequences included in the training data set. Cyclin sequences from 58 distinct organisms have been used in the training data set.(0.07 MB DOC)Click here for additional data file.Dataset S1Cyclin sequences (Positive data set) used for training the SVMs using different features.(0.06 MB DOC)Click here for additional data file.Dataset S2Non cyclin sequences (Negative data set) used for training the SVMs.(0.08 MB DOC)Click here for additional data file.Dataset S3Independent sequences (Independent data set) used for blind-test performance.(0.05 MB DOC)Click here for additional data file.


The present paper reviews the currently available data and outlines suggestions from a panel of Canadian oncologists about the use of trastuzumab and other her2-targeted agents in the neoadjuvant setting.Women receiving neoadjuvant systemic therapy for primary operable or inoperable breast cancer can potentially benefit in a number of ways, but the main advantage, which has been consistently demonstrated, is improved tumour resectability. Given the improvement in outcomes with the adjuvant use of trastuzumab in patients with early-stage breast cancer positive for the human epidermal growth factor receptor 2 (The panel focussed onher2-targeted agents as neoadjuvant therapy in primary operable, locally advanced, and inflammatory breast cancer; andthe use of trastuzumab and other possible choices of chemotherapeutic regimens with trastuzumab.her2-targeted agents emerge.The suggestions described here will continue to evolve as data from current and future trials with trastuzumab and other  A meta-analysis of randomized trials comparing neoadjuvant with adjuvant chemotherapy found no difference in distant disease recurrence  her2-positive disease observed a relative reduction in relapse of 47% (absolute reduction: 7.1%) and a relative reduction in mortality rate of 48% (absolute reduction: 2.5%) after a follow-up of 2–3 years.Trastuzumab is now the standard of care in the adjuvant setting. A meta-analysis her2- targeted agents in neoadjuvant therapy for both primary operable and primary inoperable her2- positive breast cancer is growing, and the present paper reviews the current evidence and outlines suggestions from a panel of Canadian oncologists about the neoadjuvant use of trastuzumab and other her2-targeted agents.The body of studies evaluating the use of her2-targeted agents in the neoadjuvant, adjuvant, and metastatic disease settings. Based on trial information, suggestions for each setting were formulated. For neoadjuvant therapy, a draft manuscript reviewing the currently available data and outlining the suggestions from the panel was initially written by a medical writer (BW) and reviewed and revised by four panel members . The final manuscript was reviewed and approved by the rest of the panel members . Recent clinical trial results (available as of June 2009) were incorporated into the present document, and the conclusions discussed were updated as additional evidence became available.The authors of this paper met in Toronto for a 1-day conference in June 2008. The panel reviewed the results of the latest trials using trastuzumab and other Development of the Canadian advisory panel and the present manuscript were funded by an unrestricted educational grant from Hoffmann–La Roche Canada. The authors received an honorarium for attending the conference, but not for writing the manuscript. The authors are responsible for the content of the manuscript, with no restrictions set by the sponsor.The panel focussed onher2-targeted agents as neoadjuvant therapy in primary operable, locally advanced, and inflammatory breast cancer; andthe use of trastuzumab and other possible choices of chemotherapeutic regimens with trastuzumab.The related clinical evidence is presented and discussed below. Key panel suggestions are followed by a discussion of the supporting evidence, the panel’s rationale for the suggestions, and any points on which consensus was lacking.her2-targeted therapy in the neoadjuvant setting  on days 1 and 4, 75 mg/m2 epirubicin on day 1, 500 mg/m2 cyclophosphamide on day 1] every 3 weeks for 4 cycles as neoadjuvant therapy without (p→fec) and with [h+(p→fec)] concurrent trastuzumab  ii–iiia carcinoma of the breast. The primary objective was to demonstrate a doubled improvement in pcr—defined as no evidence of residual invasive cancer in both breast and axilla—with the addition of trastuzumab to chemotherapy. The trastuzumab-based treatment arm displayed a pcr improvement by a factor of 2.5 relative to chemotherapy alone . As a result of the superior response attributed to trastuzumab, the data analysis was presented to the MD Anderson Data Monitoring Committee. Based on recommendations by that committee, the protocol of the original study was changed to discontinue the chemotherapy-alone arm. Recruitment was continued in the h+(p→fec) arm only. A total of 45 patients received the h+(p→fec) regimen. The pcr rate for the updated combined trastuzumab treatment arm (n = 45) was also superior to chemotherapy alone  was 65% at baseline in the both trial arms; after 6 months, the median lvef was 65% (range: 55%–70%) in the (p→fec) arm, but 60% (range: 52%–70%) in the h+(p→fec) arm. No clinical cardiac dysfunction was reported; however, the sample size was small, and the follow-up duration was short.In a small, two-arm, phase iii randomized noah (Neoadjuvant Herceptin) trial compared the efficacy of doxorubicin (60 mg/m2) and paclitaxel (150 mg/m2) every 3 weeks for 3 cycles, followed by paclitaxel alone (175 mg/m2) every 3 weeks for 4 cycles, followed by cyclophosphamide (600 mg/m2), methotrexate (40 mg/m2), and 5fu (600 mg/m2) every 4 weeks on days 1 and 8 for 4 cycles without (ap→p→cmf) and with [h+(ap→p→cmf)] concurrent trastuzumab  as first-line therapy in patients with her2-overexpressing labc (including ibc) her2-negative third arm was also accrued as reference for the her2-overexpressing arms, but these patients were not given trastuzumab. Data presented here refer to the group of her2-positive patients (trastuzumab arm vs. no trastuzumab arm).The international multicentre phase efs), which was defined as progression on therapy, relapse after surgery, or death from any cause. The secondary endpoints were pcr rate, overall survival, and safety and tolerability. In both her2-positive arms (with and without trastuzumab), 42%–43% of the patients presented with T4 noninflammatory breast cancer, 27% presented with ibc, and 30%–31% presented with N2 disease. In the intention-to-treat population (n = 227), patients receiving trastuzumab (n = 115) experienced a significantly improved 3-year efs rate relative to chemotherapy alone , was 34% (11/32) as compared with 68% (23/34) in the trial that gave initial lapatinib. It must be noted that, because both trials used trastuzumab with docetaxel, the lapatinib trial was not a lapatinib-only design. Further study results showed that tumours with low pten predicted resistance to trastuzumab.Recently, Migliaccio www.clinicaltrials.gov, we found 14 randomized clinical trials for her2-positive breast cancer in the neoadjuvant setting  is currently examining the safety and efficacy of the various combinations of trastuzumab and pertuzumab, another monoclonal antibody that targets acosog) Z1041 trial is examining the sequence of fec75–paclitaxel plus trastuzumab, in which fec75 is given either before or after the paclitaxel and trastuzumab. Other trials are testing new agents such as everolimus.The American College of Surgeons Oncology Group  will randomize 2547 patients to six arms: one without neoadjuvant targeted therapy, one with bevacizumab, one with only trastuzumab and no chemotherapy, one with everolimus, one with chemotherapy plus trastuzumab, and one with chemotherapy plus lapatinib. These large clinical trials may answer many clinical questions, such as “Is it better to give a combination of targeted agents rather than one agent?”her2-overexpressing breast cancer when a clinical decision has been made to treat with neoadjuvant chemotherapy. Other her2-targeting agents such as lapatinib and pertuzumab should be given only as part of a clinical trial.Currently, the panel felt that trastuzumab is standard in the neoadjuvant setting in combination with chemotherapy in patients with cr in primary operable breast cancer, and a growing body of clinical evidence shows a similar result in labc and ibccr after preoperative therapy has been shown to be a surrogate marker for long-term disease-free survival and overall survival in some trials nsabp B27 trial, despite increased pcr, no survival advantage was shown with the addition of docetaxel to an anthracycline-based regimen. Investigation of new agents in combination with conventional systemic therapies in the preoperative or neoadjuvant setting can provide tissue sampling to assess markers of in vivo tumour responses. As well, pcr can be used as a surrogate, allowing for testing of new interventions in smaller phase ii trials before they are studied in larger phase iii clinical trials, which require a larger sample size and longer follow-up.The addition of trastuzumab in neoadjuvant systemic therapy clearly increases pnoah trial. Here, the addition of trastuzumab to chemotherapy in her2-positive patients significantly improved event-free survival at 3 years relative to chemotherapy alone , with an overall survival trend  hr and absolute benefit for the addition of trastuzumab are in the range seen during the large adjuvant trials with trastuzumab; however, these results were delivered with a much smaller number of patients . The acceptable cardiac safety data also show that it may be reasonable to use a low cumulative dose of anthracycline concurrently with trastuzumab. Altogether, the noah trial has demonstrated that neoadjuvant trastuzumab for 1 year in combination with chemotherapy is a standard of care for women with labc or ibc her2-positive breast cancer. Publication of final results is eagerly awaited.The latest and strongest evidence relies on the final study results of the ncctg 9831 trial has examined the benefits of trastuzumab concurrent with chemotherapy versus trastuzumab sequential after chemotherapy. Results of the data analysis are pending. In regard to duration, based on the adjuvant trials, total duration is 1 year at present. Studies to determine if a 6-month treatment is equivalent to a 12-month treatment  or if 2 years’ treatment provides further benefits over 1 year’s treatment (hera) are ongoing. In Finland, a trial is also comparing 9 weeks with 51 weeks of adjuvant trastuzumab (NCT00593697).No trial has tested, or is testing, the effect of starting trastuzumab in the neoadjuvant setting against starting it after surgery. The Currently, the panel felt that concurrent trastuzumab with the taxane component of commonly-used neoadjuvant regimens is a reasonable standard in primary operable, locally advanced, or inflammatory breast cancer. However, current data are not strong enough to support the concurrent use of trastuzumab with anthracyclines, given the potential cardiac toxicity.labc or ibc to support any particular trastuzumab-based regimen. Several published regimens included concurrent anthracyclines with trastuzumab hera trial, higher cumulative doses of anthracyclines were found to contribute as a risk factor for trastuzumab-associated cardiotoxicity TOPO2A gene alterations, found mainly in her2-positive breast cancers The panel noted that no randomized trials were available in labc and ibc, there is increasing evidence that her2-targeted strategies used in the adjuvant setting may be applicable in the neoadjuvant setting. Data from the two randomized controlled trials of trastuzumab versus no trastuzumab in operable breast cancer  or in labc and ibc  showed that trastuzumab is associated with a significant increase in pcr. In the absence of data from randomized trials comparing neoadjuvant with adjuvant trastuzumab, the panel felt that initiating trastuzumab preoperatively and continuing it postoperatively for 1 year in total is reasonable. In an ongoing phase iii trial in her2-positive breast cancer (Neo-Adjuvant Lapatinib and/or Trastuzumab Treatment Optimisation), the total duration of her2-targeted therapy is also 1 year.In the context of primary operable breast cancer, in which patients seek a better chance for breast conservation, the panel agreed that all adjuvant data could be extrapolated to neoadjuvant therapy for clinical benefit, but with a higher chance of breast conservation. In the context of ncctg 9831 trial in which trastuzumab was given concurrently with paclitaxel in one arm and trastuzumab was given sequentially in another arm in the adjuvant setting.Whether trastuzumab should be given concurrently with chemotherapy or after chemotherapy remains an open question. Answers will be obtained with the analysis of the ac), fac, and fe60c, it is reasonable to believe that other chemotherapy regimens would be as efficacious if given preoperatively compared with postoperatively. However, the optimal duration of chemotherapy is unknown. For example, the preliminary results of the nsabp 30 trial in operable breast cancer showed that 8 cycles of chemotherapy (ac followed by docetaxel) were superior to 4 cycles of doxorubicin and docetaxel or of docetaxel, doxorubicin, and cyclophosphamide The transfer to the neoadjuvant setting of regimens proven efficacious in the adjuvant setting is supported by a meta-analysis Although the efficacy results are quite convincing, the panel warned that a decision to give concurrent neoadjuvant anthracyclines with trastuzumab could not be made based on the neoadjuvant trials conducted to date. More studies and longer follow-up from the aforementioned trials, with particular attention to cardiac effects, are required to provide further insight into the cardiac safety of such combinations.her2-positive breast cancer. The evolving data in the neoadjuvant setting suggest that the addition of trastuzumab to neoadjuvant systemic therapy significantly increases pcr, breast conservation rates, and event-free survival. A strategy of concurrent anthracyclines with trastuzumab appears promising, possibly further maximizing the efficacy of trastuzumab in high-risk patient populations. However, the cardiotoxicity risk  continues to be a concern. The present article provided panel suggestions based on available data about the use of trastuzumab preoperatively. The suggestions set out here will continue to evolve as data and experience with trastuzumab and other her2-targeted agents in trials of neoadjuvant systemic therapy emerge.Trastuzumab has become a standard of care in the adjuvant and metastatic settings for patients with Panel suggestions were completed in June 2009.


P=0.07 log-rank test, hazard ratio=1.23 ). This survival benefit was maintained over a further 2 years of follow-up ). The median survival was 138 days for placebo and 160 days for marimastat, with 2-year survival of 3% and 9% respectively. A significant survival benefit was identified at study completion in the pre-defined sub-group of 123 patients who had received prior chemotherapy ). This benefit increased with 2 years additional follow-up ), with 2-year survival of 5% and 18% respectively. Progression-free survival was also significantly longer for patients receiving marimastat compared to placebo ). Marimastat treatment was associated with the development of musculoskeletal pain and inflammation. Events of anaemia, abdominal pain, jaundice and weight loss were more common in the placebo arm. This is one of the first demonstrations of a therapeutic benefit for a matrix metalloproteinase inhibitor in cancer patients. The greatest benefit was observed in patients who had previously received chemotherapy. A further randomised study of marimastat in these patients is warranted.This randomised, double-blind, placebo-controlled study was designed to evaluate the ability of the orally administered matrix metalloproteinase inhibitor, marimastat, to prolong survival in patients with non-resectable gastric and gastro-oesophageal adenocarcinoma. Three hundred and sixty-nine patients with histological proof of adenocarcinoma, who had received no more than a single regimen of 5-fluorouracil-based chemotherapy, were randomised to receive either marimastat (10 mg b.d.) or placebo. Patients were treated for as long as was tolerable. The primary endpoint was overall survival with secondary endpoints of time to disease progression and quality of life. At the point of protocol-defined study completion  there was a modest difference in survival in the intention-to-treat population in favour of marimastat  86, 1864–1870. doi:10.1038/sj.bjc.6600310www.bjcancer.comCancer Research UK© 2002  These enzymes play an important role in normal processes of growth, differentiation and repair. The activity of MMPs is tightly regulated at several levels including gene expression and inhibition by tissue inhibitors known as TIMPs. There is now considerable evidence however, that aberrant MMP expression contributes to the invasive growth and spread of a variety of solid malignancies . MMP-2 (50s against purified enzymes in the low nanomolar range (Marimastat (BB-2516) is a broad spectrum, low molecular weight MMP inhibitor with ICSupport for the current study came from the results of a phase I trial of marimastat in patients with advanced gastric cancer . Adequate baseline hepatic function , alanine transaminase (ALT) <1.5 times the upper limit of normal) and adequate renal function  were also required. Patients were excluded if they had received radiotherapy, more than one course of systemic anti-cancer therapy, any previous investigational agent, hormonal anti-neoplastic therapy, or prior exposure to a metalloproteinase inhibitor.Patients were required to be aged over 18 years and have an ECOG performance status of 0 or 1. Patients had to have adequate bone marrow reserve at study entry,  or matched placebo in a ratio of 1 : 1 by means of a minimisation program using the following criteria: centre, gender, prior chemotherapy (yes/no), chemotherapy regimen, response to chemotherapy, performance status, extent of disease at baseline , and prior non-curative surgery (yes/no). The minimisation program also contained a random element in the event of tied minimisation scores for assigning treatments .Marimastat or matched placebo were administered in a double blind fashion as 10 mg capsules. If musculoskeletal toxicities developed, treatment was omitted until the symptoms had abated. Patients could then restart treatment at the same dose. If patients experienced a recurrence of their symptoms, treatment was again omitted until the symptoms had abated and treatment could then be restarted once daily (i.e. a 50% dose reduction). If the symptoms recurred again further treatment was at the physician's discretion, after consideration of the risk/benefit ratio for the individual patient. Once a dose reduction had been mandated, no escalation to the previous level was permitted. Patients on treatment were seen at 6 weeks, and then every 3 months, or at early termination for any reason. Treatment assignments were kept in sealed envelopes at British Biotech. The blind was only broken once all data had been verified, collected and quality assured, and the statistical analysis plan had been finalised and signed.vs placebo), with at least 90% power and using α=0.05 (two-tailed log rank test). The prospectively defined analysis point for the study was when 85% of either group had died or 18 months after the last patient was recruited, whichever occurred sooner. This point was reached in January 1999 with 85% mortality in the placebo arm. A further 2-years of survival follow-up have also been obtained up to January, 2001.The primary study endpoint was overall survival in the intention-to-treat population. A secondary analysis of overall survival in the sub-group of patients who had received chemotherapy was also pre-defined in the statistical analytical plan, since this sub-group might differ in response or outcome from the chemonaive patients. Progression-free survival, quality of life and safety and tolerability were defined as secondary endpoints. The original sample size for this study was 150 per arm. This was increased to 180 per arm when it became apparent that a higher than expected proportion of patients were leaving the study due to early disease progression (within 8 weeks of entry). The revised sample size was estimated to be sufficient to show a statistically significant difference in survival between the marimastat and placebo groups, assuming population mortality rates at 18 months of 85% for placebo and 72% for marimastat  with a 95% confidence interval using a Cox proportional hazard model containing only treatment. Quality of life data were analysed using a Wilcoxon rank-sum test and proportions were analysed using a χThe primary efficacy endpoint in this study was survival. Treatment continued until death, disease progression or drug toxicity that warranted removal from the study. Patients could receive salvage chemotherapy or other conventional anti-cancer therapy once they had withdrawn from the study.Time to disease progression was defined as the time from minimisation to documented disease progression . Progressive disease was defined as a >25% increase in the sum of the products of the largest perpendicular diameters of all measurable lesions from the study nadir. If lesions were not bi-dimensional then an unequivocal worsening of any evaluable lesion as determined by more than one investigator, the appearance of new lesions or death would constitute progressive disease. CT scans were performed at baseline and thereafter at 3 monthly intervals or at early termination if there was clinical suspicion of relapse. Patients dying prior to documented progressive disease were considered to have experienced progressive disease at death. Quality of life was measured by the QLQ-C30 questionnaire at screening, weeks 6 and 12 and every 3 months thereafter up to 36 months.Medical history was recorded at baseline. Performance status, full blood count and serum chemistry profile were recorded at baseline, after 6 weeks, 3 months and 3 monthly thereafter up to 18 months. All signs, symptoms and laboratory abnormalities were assessed using the NCI CTC criteria for toxicities. In addition, a specific rating for grading musculoskeletal toxicity was developed for this study. Grade 1 for musculoskeletal toxicity was defined as aches and pains with no restriction of activity. Grade 2 was defined as having pain causing restriction of activity. Grade 3 was defined as having pain and the presence of nodules or clinically inflamed joints or tendons. Grade 4 was defined as pain and the presence of a contracture.A total of 369 patients were recruited from 37 European hospitals between October 1996 and October 1998. Of these, 185 received marimastat and 184 received placebo. One patient in the marimastat arm was lost to follow-up. The baseline patient demographics are shown in Table 1Seventeen patients were deemed to be protocol deviators, 10 in the marimastat group and seven in the placebo group. Two marimastat patients and one placebo patient had potentially curative resections and entered the study without histological or radiological confirmation of residual or relapsed disease. Two marimastat patients and one placebo patient had received prior radiotherapy. Previous malignant disease was recorded for one marimastat patient (testicular cancer) and two placebo patients . Six marimastat patients and three placebo patients showed signs of disease progression between completion of first-line treatment and study entry, and did not take any study drug even though they were minimised. In addition, six marimastat patients and three placebo patients entered the study with disease that would normally be considered resectable . However, a combination of advanced age and overall frail condition precluded the possibility of resection. Although technically these patients did not deviate from the protocol they may be considered to have introduced an imbalance in the arms and this is explored in the following section.−1 at week 6 (n=147) and 49.2 ng ml−1 at week 12 (n=101). Due to variability in sampling times these values cannot be regarded as true troughs. The concentrations were in line with expectations from previous studies and equate to free-drug concentrations of approximately 3–4 times the IC50 for the target enzymes such as gelatinase A and MT1-MMP.Pharmacokinetic analysis revealed mean plasma concentrations of marimastat of 67.6 ng mlP=0.043 log-rank test, HR=1.27 (1.00–1.60)), but failed to meet the endpoint when all patients are included (ITT) ). The survival difference in favour of marimastat was maintained with a further 2 years of follow-up ), with median survival times of 160 days for marimastat and 138 days for placebo, and 2-year survival of 9% and 3% respectively  for result after 2 years of additional follow-up.The primary analysis was performed when 85% of patients in the placebo arm had died. The primary endpoint of the study was met when the nine patients who did not take the study drug were excluded (modified ITT) ). This survival difference increased with 2 years of additional follow-up ), with median survival times of 253 days for marimastat and 175 days for placebo, and 2-year survival of 18% and 5% respectively ).Analysis of overall survival in the predefined sub-group of patients who had received prior chemotherapy revealed a significant benefit for marimastat at the primary analysis point ). This difference was maintained over the 2 years of additional follow-up ) , and in favour of placebo for metastases (M1 72% vs 68%) and no prior resection (74% vs 65%). Another source of imbalance was recruitment of three placebo patients and six marimastat patients who had resectable disease . The chemotherapy sub-group contained one patient with resectable disease in each arm. When these patients were excluded, the overall survival benefit after 2 years additional follow-up was reduced and only just significant ). However, the result for the chemotherapy sub-group was largely unchanged ).The possibility of bias or imbalance between the arms was explored as an explanation for the survival differences. Interactions between several factors precluded a single multifactorial analysis to adjust for baseline prognostic factors. There was a small imbalance in ECOG status in the ITT population with 70% of placebo patients having a status of 1 The robustness of the survival benefit in the chemotherapy sub-group was explored further by Cox regression analyses to account for individual prognostic factor imbalance. None of the factors diminished the hazard ratio below 1.61, and the imbalance in prior surgical debulking increased the hazard ratio to 1.77. Furthermore, when divided in two halves on the basis of recruitment period, a survival trend in favour of marimastat is seen in both halves (HR=1.51 and HR=2.04).vs 56.5, P=0.49) or change from baseline standardised AUC  over the first 12 weeks of the study (Wilcoxon rank-sum tests). Further analysis was prevented by a marked reduction in the number of patients remaining on study and able to complete the questionnaire.Quality of life analyses using the EORTC-QLQC30 instrument were performed at baseline, 6 weeks, 12 weeks and at 3-monthly intervals thereafter. There was no statistically significant difference between placebo and marimastat in standardised area under the curve (AUC)  and two placebo patients (1%) withdrew from the study due to adverse events. Events of arthralgia, joint stiffness, limb pain, general pain, myalgia, peripheral oedema, dysphagia and fatigue were more common in the marimastat treatment arm (>5% absolute difference). The majority of these events were related to the musculoskeletal syndrome that has been described for marimastat and several other MMPIs. The condition is characterised by musculoskeletal pain and inflammation, most commonly originating in the upper shoulder girdle or hands. The condition tended to develop in the second and third months of treatment at 10 mg b.d. and in five cases led to contractures of the palm and fingers. A peripheral oedema of the hands was also sometimes seen in patients with these musculoskeletal side effects. The condition was managed by treatment interruption and many patients were able to resume treatment on the same or lower dose after a break of 2–3 weeks. Severe events could generally be avoided by early treatment interruption. A more general tiredness, recorded in a higher incidence of both fatigue and somnolence, may also be associated with marimastat treatment.Events of abdominal pain, jaundice, weight loss, anaemia, and ascites were more common in the placebo treatment arm (>5% absolute difference). The increased incidence of anaemia was also evident in a 32% increase in transfusion requirement for the placebo arm over marimastat. This is consistent with the reduction in tumour haemorrhage observed endoscopically in the phase II study of marimastat in advanced gastric cancer, and with the intended therapeutic effect of marimastat.P=0.001, χ2 test). A comparison of NCI-CTC graded laboratory abnormalities revealed an increased incidence of grade 3/4 low hemoglobin (<7.9 g dl−1) in the placebo arm   are shown for both arms in Table 3The significance of the musculoskeletal side effects of marimastat can be seen in Figure 4The primary result of this randomised trial shows a modest survival benefit for marimastat treatment in the overall population of gastric cancer patients. The more interesting result, however, is seen in the sub-group of patients who had received prior chemotherapy. The survival benefit in this group, if true, is clinically significant with an absolute 2-year survival difference of 13%, and a 45% improvement in median survival. There are obvious concerns regarding the interpretation of a result from a sub-group even if, as in this case, it was pre-defined. For this reason the robustness of the result was explored further. Cox regression analysis indicated that imbalances of individual prognostic factors were unlikely to account for the survival difference. A survival benefit in favour of marimastat was also observed in both halves of the chemotherapy sub-group when divided arbitrarily on the basis of recruitment period. Finally, the result is not significantly reduced by the exclusion of the single patient in each arm of the sub-group who entered the study with potentially resectable disease .It is important to note that the chemotherapy sub-group is a selected population, including as it does only those patients who had responded to chemotherapy or who had shown stable disease during treatment. The reason these patients responded to marimastat may be related to the fact that they responded in some way to the chemotherapy. Alternatively, the marimastat benefit may be related to the fact that this sub-group excludes those patients with more advanced or more rapidly progressive disease.The safety and tolerability of marimastat was generally good, with only those events related to the musculoskeletal syndrome being significantly elevated above placebo. The musculoskeletal side effects clearly limit the duration of treatment for the majority of patients. However, the condition can be readily monitored and can be managed by treatment interruptions. In this study 21 placebo patients and 69 marimastat patients interrupted treatment, with a median duration of 14 days for both groups. Experience in this and other studies has shown that severe musculoskeletal side effects can usually be avoided by prompt treatment breaks. It is possible that improved treatment schedules and pre-medications could be employed to allow longer term dosing.Early expectations for the use of MMP inhibitors in cancer treatment were high, but previous clinical trials with this new class of agent have been disappointing. Marimastat at a dose of 25 mg b.d. showed comparable 1-year survival to gemcitabine in patients with non-metastatic pancreatic cancer , ovarian14C-marimastat in the rat have shown very high concentrations of marimastat in the stomach wall even after washing. By contrast the levels in lung were low (British Biotech – unpublished data).Against this background the current results are encouraging. This study of marimastat in patients with gastric cancer provides the first indication of a survival benefit for an MMP inhibitor. The reason that benefit is seen with this cancer and not others may be due to inadequate tissue concentrations of the drug. Biodistribution studies with In conclusion, these results support a possible role for marimastat as a maintenance treatment following a response or stable disease to chemotherapy. 5-FU based combination chemotherapy is gaining acceptance as the standard of care for gastric and gastro-oesophageal cancer patients with reasonable performance status, both in Europe and North America. In this setting, MMP inhibitor therapy may prove to be a valuable component of the anticancer armoury, and confirmatory trials of this novel agent are warranted.


Hemodynamic instability following the changeover of vasoactive infusion pump (CVIP) is a common problem in the intensive care unit. Several empiric methods are used to achieve CVIP. We hypothesized that the variation in these procedures could generate some morbidity. We sought to assess the effects of the standardization of practice, as a quality improvement program, on the CVIP-induced incidents.We performed a prospective before-and-after intervention study including all adult patients with a diagnosis of cardiovascular failure who received a continuous infusion of vasoactive drugs or inotropic drugs. After a baseline preimplementation period (phase 1), a standardized 'quick change method' of CVIP using two syringe drivers was implemented in our intensive care unit (phase 2). Endpoints  were registered in both 3-month phases.P < 0.0001) reduced in phase 2  versus phase 1 . This effect was observed whichever catecholamine was used. More than 98% of incidents were blood pressure variations, with a similar distribution of the nature of incidents in both phases.We studied a total of 913 CVIP events  from 43 patients. Patient characteristics were not significantly different among phases, with a majority of the patients having septic shock. The frequency of incidents was significantly (The present study illustrates that adverse events are common following CVIP, and illustrates the positive impact of a quality improvement program to enhance inpatient safety related to this current process of care. Circulatory failure is one of the most common organ dysfunctions in patients admitted to the intensive care unit (ICU) . These pSeveral procedures are commonly used to achieve these infusion exchanges, using either a single syringe driver  or two sWe hypothesized that the lack of standardization could result in a greater number of adverse events related to CVIP. The aim of the present study was to assess the influence of a quality improvement program to help reduce the number of incidents linked to this current care in the ICU.Following an internal audit in our institution, we identified CVIP to be a daily problem. As recommended to overcome such difficulties -12, we dThe first prospective and observatory phase of the study was conducted over a 3-month period. All adult patients suffering from shock who received continuous infusion of catecholamines  were observed according to this quality improvement program. Cardiovascular dysfunction was defined by a systolic arterial blood pressure <90 mmHg with signs of peripheral hypoperfusion despite adequate fluid resuscitation, and by a continuous infusion of vasopressor agents or inotropic agents required to maintain systolic pressure ≥90 mmHg. In accordance with our routine clinical practices, all patients who received these drugs had a central venous catheter. Catecholamines were always infused with a minimal flow rate of approximately 2 ml/hour. Patients were continuously monitored, including their invasive arterial blood pressure. The aim of this first phase was to record all the incidents (as defined below) related to the CVIP. In this phase, nurses were free to choose the method of changeover of CVIP they usually practice.®; Fresenius Viale™, Brezin, France) was used for this study, with 50 ml syringes . The protocol was continuously available for consultation in the ICU.The second study phase was designed to standardize procedures for CVIP and to change behaviors. Following phase 1, during a 1-month period each nurse received a 1-day training course including practical work. We chose as an effective strategy the previously described 'quick change method', using two syringe drivers, already known by the staff -8. This After this training period, as in phase 1, we recorded throughout another 3-month period all incidents related to the CVIP in patients treated with catecholamines.®; Hewlett Packard™, Louisville, KY, USA). Baseline hemodynamics were defined as the average of three measurements preceding CVIP.Baseline characteristics of the patients were registered: gender, age, Simplified Acute Physiology Score II, type of admission and type of shock. Data related to CVIP were also recorded: the catecholamine and its dose. The heart rate and invasive systolic blood pressure were continuously recorded 5 minutes before and throughout CVIP on a monitor  based on prestudy observations, we calculated that at least 736 CVIP events would be required for the study to have 90% power to detect a 50% reduction in the relative risk with a two-sided α level of 5%. Data are expressed as counts and proportions or as the mean ± standard deviation, as appropriate. Comparisons of categorical variables were performed using a two-sided chi-square test or Fisher's exact test, as appropriate. Continuous data were compared using an unpaired Student's P = not significant).We studied 43 patients: 25 patients in phase 1 and 18 patients in phase 2. Characteristics of the patients are presented in Table P = not significant). The distribution of changeovers according to the catecholamines is presented in Table P < 0.05) – with more changeovers of dobutamine and dopamine in phase 2  than in phase 1 , and more changeovers of norepinephrine in phase 1 (48%) than in phase 2 (32%).From these patients, 913 CVIP events were evaluated: 435 events in phase 1 and 478 events in phase 2. The number of CVIP procedures per patient was not significantly different in phase 1 (17.4 ± 23.4) versus phase 2 (26.5 ± 22.3) (P < 0.05). The other mean concentrations of catecholamines  were not significantly different between both phases of the study.The doses and flow rates for each inotropic/vasoactive drug are presented in Table P = 0.02) reduced from phase 1 . We recorded a total of 106 incidents during the study: 78 incidents in phase 1 and 28 incidents in phase 2. The frequency of incidents was significantly lower in phase 2 than in phase 1 (P < 0.0001), at 5.9% and 17.8%, respectively. In addition, as presented in Table P = 0.18) among phases  was significantly  'quick change method' using two syringe drivers as an effective strategy to standardize our CVIP practices. Indeed, it appeared that this procedure had at least three advantages: 'the quick change method' minimized the zero-infusion time using two pumps, was very quick and simple, and was less time-consuming for nurses.Thanks to this quality improvement program in the ICU, by changing staff behaviors, the intervention reduced the occurrence of adverse events by about 67%. We observed a similar distribution of incidents in both phases of the study with a wide majority of blood pressure variations. This dramatic patient safety improvement was effective whichever catecholamine was used, including vasoactive drugs.Our positive results are all the more sound since doses of catecholamines related to most incidents  are significantly higher in the second phase of our study. After standardization, we still observed 6% CIVP-induced incidents. We can speculate that part of these incidents, related to devices and/or imperfections of the method, cannot be cut down in the setting of critically ill patients. It could be interesting, however, to compare the positive results we obtained using the 'quick change method' with those we could expect from new expensive smart pumps, with a modular design, assisted by an internal computer, which can allow automatic relays . FurtherIn summary, the present study illustrates the high morbidity rate related to CVIP in the ICU, and provides some evidence to standardize these risky procedures to improve inpatient safety. The study emphasizes also the necessity in the future for a continuous quality improvement program, including nurses in interdisciplinary teamwork, into general use of all ICU healthcare practices.• Hemodynamic instability following CVIP is a common problem in the ICU.• Without guidelines currently available, several empiric methods are commonly used to achieve changeovers of syringes.• The present study illustrates the high rate of blood pressure variations associated with the lack of standardization of these procedures.• Implementation of a standardized method, based on clinical evidence and local resources, could dramatically reduce adverse events linked to this practice.CVIP = changeover of vasoactive infusion pump; ICU = intensive care unit.The authors declare that they have no competing interests.LA conceived of the study, participated in its design and coordination, helped to draft the manuscript and performed the statistical analysis. MC participated in the analysis and interpretation of data and helped to draft the manuscript. OM, MS-D, TF and AG participated in the design of the study, in nurse training for the protocol and in the acquisition of data. DR coordinated the study, and was involved in revising the manuscript critically. All authors read and approved the final version of the manuscript.