The marked hypermuscularity in mice with constitutive myostatin deficiency reduces fat accumulation and hyperglycemia induced by high-fat feeding, but it is unclear whether the smaller increase in muscle mass caused by postdevelopmental loss of myostatin activity has beneficial metabolic effects during high-fat feeding. We therefore examined how postdevelopmental myostatin knockout influenced effects of high-fat feeding. Male mice with ubiquitous expression of tamoxifen-inducible Cre recombinase were fed tamoxifen for 2 weeks at 4 months of age. This depleted myostatin in mice with floxed myostatin genes, but not in control mice with normal myostatin genes. Some mice were fed a high-fat diet (60% of energy) for 22 weeks, starting 2 weeks after cessation of tamoxifen feeding. Myostatin depletion increased skeletal muscle mass ∼30%. Hypermuscular mice had ∼50% less weight gain than control mice over the first 8 weeks of high-fat feeding. During the subsequent 3 months of high-fat feeding, additional weight gain was similar in control and myostatin-deficient mice. After 5 months of high-fat feeding, the mass of epididymal and retroperitoneal fat pads was similar in control and myostatin-deficient mice even though myostatin depletion reduced the weight gain attributable to the high-fat diet . Myostatin depletion did not alter fasting blood glucose levels after 3 or 5 months of high-fat feeding, but reduced glucose levels measured 90 min after intraperitoneal glucose injection. Myostatin depletion also attenuated hepatic steatosis and accumulation of fat in muscle tissue. We conclude that blocking myostatin signaling after maturity can attenuate some of the adverse effects of a high-fat diet. When laboratory mice are fed a high-fat diet, they become obese and have hyperglycemia and hepatic steatosis as often observed in obese humans. This diet-induced obesity is attenuated in mice in which there is marked hypermuscularity caused by constitutive absence of functional myostatin or constitutive expression of a protein that inhibits myostatin activity ob/ob mice, administration of an anti-myostatin antibody for 6 weeks increased muscle mass by about one-third but did not attenuate the rapid increase in adiposity during this period The studies cited above raise the possibility that an anti-myostatin therapy, if one can be developed, would reduce fat mass, glucose levels, and hepatic steatosis in obese humans. However, at least in mice, the increase in muscle mass that can be achieved by blocking myostatin activity after muscle development is only about one-fourth to one-third of the increase in muscle mass caused by constitutive myostatin deficiency All procedures were approved by the University of Rochester animal research committee and were consistent with all regulations on humane use of animals for research .Mice in which exon 3 of the myostatin gene is flanked by loxP sequences (Mstn[f/f]) have been described previously When the mice were 4 months old, their usual low-fat chow  was withdrawn and replaced with low-fat chow containing tamoxifen (250 mg/kg) for a two week period to activate the CreER protein. This procedure knocks out myostatin exon 3 in Mstn[f/f]/CreER+mice th and 21st week of high-fat feeding. Blood samples were obtained in the afternoon, 5 hr after chow was removed from the cage, both before and after ip injection of glucose (1 mg/g body weight). Initially, we tested glucose concentrations at 30, 60, and 90 min after glucose injection. This frequent sampling protocol was stressful to the mice and resulted in highly variable and sometimes unexpectedly high glucose concentrations even in mice consuming normal chow. Hence, we changed the protocol to sample blood only once after the glucose injection, at 90 min. Mean fasting glucose levels reported here include data from all mice, but the mean 90 min post-injection levels are based on only those mice that had a single post-injection blood sample.Two weeks after the completion of tamoxifen administration, some of the mice were switched to a high-fat diet . Other mice continued to consume the low-fat LabDiet 5010. The mice stayed on the assigned diets for 22 weeks. They were weighed at 2 week intervals. Mandibular vein glucose concentrations were determined with a FreeStyle Freedom™glucose meter (Abbott) during the 13After 22 weeks of high-fat feeding, or the same period of low-fat feeding, mice were euthanized for determination of the mass of skeletal muscles , heart, liver, kidney, and intra-abdominal fat pads . In mice fed the low-fat diet, retroperitoneal and epididymal fat pads accounted for nearly all of the visible adipose tissue within the abdominal cavity, except for very small amounts adhering to intestine and pancreas. In mice fed the high-fat diet, adipose tissue surrounding gastrointestinal organs appeared to be continuous with the retroperitoneal adipose tissue and was classified as such. Muscles were snap frozen in liquid nitrogen and stored at −70°C. Livers were stored in 10% buffered neutral formalin. Fatty acid contents of triceps muscles, which included lipids within muscle fibers and in adipose tissue between fascicles, were determined by gas chromatography with C17∶0 as an internal standard P for multiple comparisons. The denominator for computing the t values was based on the residual variance from the ANOVA. We analyzed the high-fat and low-fat conditions separately rather than adding dietary condition as an additional factor in the ANOVA, because variance was greater for the high-fat condition. Thus, using residual errors pooled across dietary conditions would have inflated the statistical significance of the effects of myostatin deficiency during high-fat feeding. Variance was higher with the high-fat diet for several other outcome measures, including fat pad mass, liver mass, glucose levels after ip glucose injection, and intramuscular lipid content. We therefore assessed the effects of myostatin depletion separately for the low-fat and high-fat conditions rather than performing ANOVA with diet as a factor. P values for effects of myostatin depletion within diet groups were determined with t-tests. Two-tailed P values are reported. For simplicity and consistency, this approach was taken for all outcome measures even when variance was similar in low-fat and high-fat conditions. The statistical significance of differences in outcome measures for high-fat feeding versus low-fat feeding in both myostatin-deficient and control mice were determined by t-tests adjusted for unequal variance between groups. Fisher's exact probability test (two-sided) was used to determine whether the distribution of steatosis scores in mice fed the high-fat diet was significantly different in control and myostatin-deficient mice. R version 2.12.0 was used for ANOVA and Fisher's test, and Microsoft Excel was used for t-tests.Data are expressed as means, and where error terms are shown these are standard errors of the mean. Factorial analysis of variance  was done to assess the statistical significance of the effect of myostatin depletion on biweekly weight changes and biweekly cumulative weight gain. The statistical significance of the effect of myostatin depletion at each time point was assessed by Bonferroni t-tests to adjust P<0.01). Mice of both genotypes lost some body mass during 2 weeks of tamoxifen feeding . During the first 2 weeks after tamoxifen feeding, before the mice started consuming the high-fat diet, Mstn[f/f] mice gained more weight than Mstn[w/w] mice , presumably because of a rapid increase in muscle mass . Thus, body mass was greater (P<0.001) in myostatin-depleted mice at the onset of high-fat feeding (29.1 vs. 26.5 g) and at the same time point in mice that continued on the low-fat diet (30.1 vs. 27.0 g). After the more rapid weight gain in Mstn[f/f] mice during the first 2 weeks post-tamoxifen, there was no further effect of myostatin depletion on the increment in total body mass in mice in which the low-fat diet was continued during the final 22 weeks of the study .Before tamoxifen administration, the mean body mass of Mstn[f/f] mice was slightly greater than that of Mstn[w/w] mice, 27.6 vs. 26.1 g (P<0.05 for myostatin status×time interaction). The only individual time interval with significantly lower weight gain (P<0.05) in myostatin-deficient mice was the interval between 2 and 4 weeks after starting the high-fat diet. Myostatin-deficient mice had a greater body mass when the high-fat diet was started (because of increased muscle mass), so their smaller weight gains did not result in significantly lower body mass at any time point . Myocardial mass was increased by the high-fat diet, 14% in control mice and 10% in myostatin-deficient mice. Kidney mass was not significantly affected by the high-fat diet in either normal or myostatin-deficient mice .The high-fat diet induced an increase of ∼4-fold in the amount of fat in triceps muscles in mice with normal myostatin levels . In contMice made hypermuscular by postdevelopmental myostatin depletion gained less weight than normal mice during the first few weeks of exposure to a very-high-fat diet. However, the further increase in weight beyond the first few weeks of high-fat feeding was not significantly affected by myostatin depletion. After 5 months of high-fat feeding, the intra-abdominal fat mass was similar in normal and myostatin-deficient mice. The mean total weight gain associated with high-fat feeding  was 29% less in myostatin-deficient mice, but we could not account for this difference based on the fat depots and organs that were weighed in this study. Subcutaneous adipose tissue has a very large capacity to store fat and a difference in subcutaneous fat accumulation is a potential explanation for the differential weight gain. Unfortunately, we did not include measurements of subcutaneous fat mass when designing the study because previous research indicated that myostatin knockout or inhibition reduced epididymal, retroperitoneal, and subcutaneous fat mass to the same extent ob/ob mice, in which the increment in muscle mass caused by myostatin blockade was very similar to the increment in muscle mass induced by myostatin depletion in the present study Previous research has demonstrated that inhibition or deficiency of myostatin does not reduce, and may increase, intake of normal or high-fat chow In mice with constitutive expression of a protein that inhibits myostatin activity, a high-fat diet fed during development (4–23 weeks of age) increased body mass and muscle mass more in myostatin-deficient than in mice with normal myostatin activity Presumably, most of the increase in liver mass associated with high-fat feeding was caused by steatosis, which was evident in the histological examinations of liver sections. The mean increase in liver mass in the obese myostatin-deficient mice (<0.1 g) was markedly attenuated compared with that of the obese mice with normal myostatin expression (0.5 g), and histology indicated that steatosis was not as severe in the myostatin-deficient mice. A constitutive myostatin gene mutation also attenuated the hepatic steatosis induced by a high-fat diet ob/ob mice treated with an anti-myostatin antibody had reduced glucose levels when consuming normal low-fat food, but this occurred without a change in the glucose-lowering effect of exogenous insulin After a period of fasting, when the serum insulin level is low, muscle consumes fat rather than glucose as its primary fuel source. Thus, it is not surprising that the hypermuscularity in myostatin-deficient mice did not reduce glucose levels after a 5 hr fast. When insulin levels are elevated, muscle becomes a major glucose consumer. The lower glucose levels in myostatin-deficient mice 90 min after glucose administration could simply reflect the increased mass of an insulin sensitive tissue. It also is possible that loss of myostatin increased sensitivity to insulin. Although insulin resistance has been induced by injecting myostatin into mice All research in this area, including the present study, has examined whether lack of myostatin activity attenuates the development of obesity or hyperglycemia when myostatin activity was knocked out or inhibited before the mice became obese. Future studies should be designed to determine whether inhibition of myostatin signaling can help to reverse established obesity, hepatic steatosis and glucose intolerance.


To examine how symptom cluster subgroups defined by extreme discordant composite scores, cut-off scores, or a median split influence statistical associations with peripheral cytokine levels in women with breast cancer.Systemic cytokine dysregulation has been posited as a potential biological mechanism underlying symptom clusters in women with breast cancer. Symptom characteristics may play an important role in identifying cytokines of significant etiological importance, however, there is no consensus regarding the ideal subgrouping technique to use.A secondary analysis of data collected from a cross-sectional descriptive study of women with stage I-II breast cancer was used to examine and compare the relationships between peripheral cytokine levels and symptom subgroups defined by extreme discordant composite scores, cut-off scores, or a median split.Participant symptom scores were transformed into a composite score to account for variability in symptom intensity, frequency and interference. Cytokine levels in subgroups defined by composite scores within the highest and lowest 20% were contrasted with those composed from cut-off scores and a median split.Subgroups defined by the composite score or cut-off scores resulted in similar statistical relationships with cytokine levels in contrast to the median split technique. The use of a median split for evaluating relationships between symptoms clusters and cytokine levels may increase the risk of a type I error.Composite and cut-off scores represent best techniques for defining symptom cluster subgroups in women with breast cancer. Using a consistent approach to defining symptom clusters across studies may assist in identifying relevant biological mechanisms. More than 220,000 women will receive a diagnosis of breast cancer (BC) this year in the United States . While aAlthough there is ample evidence showing a high prevalence of symptoms clusters among women with BC and negative effects on quality of life, there have been few studies focused on exploring potential biological mechanisms associated with these symptoms. Identification of the biological mechanisms underlying the development and persistence of this symptom group, known as the psychoneurological (PN) symptom cluster, could lead to more targeted symptom management strategies .A putative mechanism of PN symptoms in women with BC involves ongoing inflammation resulting from dysregulation of the hypothalamic-pituitary adrenal (HPA) axis. Support for the connection between HPA axis dysfunction, inflammation and symptom clusters is derived from the research describing cytokine-induced sickness behavior in animal models and human studies . The sicAlthough there are some evidences that supports a relationship between PN symptoms and alterations in neuroendocrine hormones and proinflammatory cytokines in women with BC, the findings have not been consistent ,18. ThisIn contrast, homogeneity of the symptom cluster subgroup can be increased by using symptom domains  to identify subgroups with similar symptom characteristics. For instance, among 191 individuals with cancer receiving treatment, PN symptom clusters based on symptom severity revealed four subgroups—all low symptoms, high pain low fatigue, high fatigue low pain, all high symptoms . This meProviding a basis to distinguish those who definitely have the symptom cluster from those who definitely do not, an extreme discordant analytic approach can be used to increase statistical power to detect relevant biological mechanisms. This is similar to the sampling designs used by researchers in genetic epidemiology that are used to discover causal variants . By seleA secondary analysis of 128 women with BCA was subsequently undertaken to contrast the application of extreme discordant subgroups defined by composite scores, cut-off scores or a median-split. The dataset was part of a larger research study carried out in two university health systems in the mid-Atlantic region from 2004-2009. Women diagnosed with Stages I-II BCA were approached about study participation at 4 weeks after lumpectomy or mastectomy, before receiving their first dose of chemotherapy. This time point was selected because it provided an adequate duration of time after surgery for return of baseline immune parameters.i.e. multiple sclerosis, HIV, lupus), use of anxiolytics or anti-depressants, or regular use of anti-inflammatory medications. All participants verbalized understanding and gave informed consent to the research protocol, which was approved by the university's institutional review board.Inclusion criteria were women over the age of 18 years diagnosed with Stages I-II BCA after lumpectomy or mastectomy and fluency in English. Exclusion criteria included a past medical history of cancer or immune-related disease  to severe (4) . A totalPlasma concentrations of cytokines were measured with the Bio-Plex Human 17-Plex . This standardized kit includes coupled beads, detection antibodies, and standards for the detection of IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, G-CSF, granulocyte-macrophage colony-stimulating factor, interferon-gamma (IFN-γ), monocyte chemoattractant protein-1, macrophage inflammatory protein-1β, and TNF-α. After incubation, contents of each microplate well were drawn into the Bio-Plex array reader, and precision fluidics align the beads in a single file through a flow cell, where two lasers excite the beads individually. High-speed digital signal processors and Bio-Plex Manager software  record the fluorescent signals simultaneously for each bead. Levels of CRP were determined using a high-sensitivity enzyme-linked immunosorbent assay (ELISA) assay . Sensitivity of all measurements = 10 pg/mL.Cut-off scores for each symptom assessment measure were derived from the literature and were correlated with moderate to severe symptom intensity, frequency, and interference. A cut-off score of 6 or above was applied to indicate moderate fatigue, measured on the Symptom Experience Survey. Depressive symptoms were cut-off score at ≥16, the traditional indicator for probable depression on the CES-D. A cut-off score of 6 or above was applied to indicate moderate sleep disturbance, measured on the Symptom Experience Survey. A cut-off score of ≥4 was used for the Brief Pain Inventory-Short Form, indicating moderate pain severity and interference. However, there were only three participant scores that met the cut-off scores applied to pain and sleep disturbances. Because the sample scores for pain and sleep disturbances were generally low, the analysis proceeded with the symptom cluster of fatigue and depressive symptoms. Participants with both symptom scores above the cut-off were designated as the high-symptom cluster group while participants with both symptom scores below the cut-off were designated as the low-symptom cluster group.n = 25) with the highest composite symptom cluster score were selected. Of these, 20/25 (80%) met the cut-off scores for fatigue and depression, 25/25 (100%) met the cut-off for depression, and 20/25 (80%) met the cut-off score for fatigue. In contrast, when using the cut-off scores for fatigue and depression as inclusion in the extreme symptom cluster phenotype, there were 24 participants who fell within this group (blue line).To standardize symptom scores among the instruments used, the symptom scores were transformed to a 1 - 100 scale. This transformation provided equally weighted scales so that each score value contributed equally to the sum score. A composite symptom cluster score was computed for each individual by summing the two transformed scores. The subgroup of women with the highest composite symptom cluster score, defined as the top 20%, were chosen for the extreme discordant subgroup analysis. In the same manner, the 20% of women with the lowest composite symptom score comprised the low symptom cluster group. With a sample of 128 women with BCA, the top 20%  or cluster of symptoms (fatigue and depression) results in extreme discordant subgroups, which can maximize statistical power. Based on this model, cytokine profiles were examined and compared using the composite symptom cluster score, symptom cut-off scores, and a median split of the sample.SD 7.7, Range 27 - 63), was mainly Caucasian (62.5%) and was slightly more post-menopausal (53%) than pre-menopausal (46.9%). The majority of women underwent mastectomy (65.6%) as opposed to lumpectomy (34.4%).This sample of 128 women had a mean age of 47.7 years  in the total score. The step of transforming each symptom scale to 0 - 100 composite score allowed equal representation of each domain as well as each symptom in the composite score. Measuring multiple domains of the symptom is consistent with a patient-centered approach to symptom measurement as a high intensity does not always translate to high interference. In contrast with the composite score technique, the cut-off score resulted in the inclusion of some symptom data that was less dependent while excluding others that were highly dependent. The median split technique resulted in the highest amount of variability in symptom scores and levels of significance in cytokines, suggesting this approach may increase the risk of Type I errors.With an increasing focus of research on identifying the biological basis of symptom clusters in women with breast cancer, it would be advantageous to have one definition of a symptom cluster that is implemented across studies. Using a composite symptom cluster score or cut-off score to identify subgroups is an alternative until consensus on a precise definition of a symptom cluster can be reached. Both composite and cut-off score techniques provide a way to identify and compare women who have the most severe with the least severe symptoms, which can increase the likelihood of detecting clinically significant biological mechanisms.Research studies focused on identifying clinically relevant biological mechanisms of symptom clusters experienced by individuals with cancer often use an a priori approach to symptom assessment. It is imperative to initially assess the range and distribution of each symptom score of the study sample to determine if the symptom cluster is indeed observed in the study sample. The best means for identifying subgroups is through using a composite score or assigning a clinically relevant cut-off score, so that the analysis may be performed among groups with the most similar symptom pattern.


Angiogenesis is regarded as a hallmark in cancer development, and anti-angiogenic treatment is presently used in non-small cell lung cancer (NSCLC) patients. MicroRNAs (miRs) are small non-coding, endogenous, single stranded RNAs that regulate gene expression. In this study we aimed to identify significantly altered miRs related to angiogenesis in NSCLC.in situ hybridization (ISH) in the total cohort (n = 335) and correlation analyses with several well-known angiogenic markers were done.From a large cohort of 335 NSCLC patients, paraffin-embedded samples from 10 patients with a short disease specific survival (DSS), 10 with a long DSS and 10 normal controls were analyzed. The miRs were quantified by microarray hybridization and selected miRs were validated by real-time qPCR. The impacts of different pathways, including angiogenesis, were evaluated by Gene Set Enrichment Analysis (GSEA) derived from Protein ANalysis THrough Evolutionary Relationship (PANTHER). One of the most interesting candidate markers, miR-155, was validated by 128 miRs were significantly up- or down-regulated; normal versus long DSS (n = 68) and/or normal versus short DSS (n = 63) and/or long versus short DSS (n = 37). The pathway analysis indicates angiogenesis-related miRs to be involved in NSCLC. There were strong significant correlations between the array hybridization and qPCR validation data. The significantly altered angiogenesis-related miRs of high interest were miR-21, miR-106a, miR-126, miR-155, miR-182, miR-210 and miR-424. miR-155 correlated significantly with fibroblast growth factor 2 (FGF2) in the total cohort , though most prominent in the subgroup with nodal metastasis .Several angiogenesis-related miRs are significantly altered in NSCLC. Further studies to understand their biological functions and explore their clinical relevance are warranted. Lung cancer is the number one cause of cancer related mortality in both men and women, and approximately 80% are non-small cell lung cancer (NSCLC) MicroRNAs (miRs) are small non-coding, endogenous, single stranded RNAs that regulate gene expression Some miRs appear to be involved in angiogenesis The first evidence showing miRs in the regulation of angiogenesis came from Dicer knockout mice. Dicer is a critical enzyme in miR synthesis. The Dicer-deficit mice died early during development due to the thinning of vascular walls and severe disorganization of the network of blood vessels in situ hybridization In a large unselected NSCLC cohort we have previously studied the prognostic impact of several key angiogenic growth factors and receptors as well as the prognostic role of miR-155 and miR-126 by Demographic, clinical, and histopathological variables in the patient groups  are shown in Out of 281 miRs, Scatter plots comparing microarray hybridization and qPCR data from 28 selected miRs are shown in in situ hybridization (ISH) analysis of NSCLC representing strong and weak intensities for tumor cell miR-155 expression as well as negative and positive controls. We have previously published on the prognostic impact of miR-155 in our large (335 patients) NSCLC population To our knowledge, this is the first NSCLC miR expression profiling study showing a gene set connected to angiogenesis-related miRs with the highest impact in pathway analysis. Consistent with previously published studies we observe significant differences in miR profiles between normal and tumor tissue and between samples from patients with a poor versus a favorable DSS. Several of these significant altered miRs are associated with angiogenesis and are interesting candidate markers for further evaluation. By validating one of these markers, miR-155, in a cohort of 335 NSCLC patients, we find this miR for the first time to be significantly associated with the well-known angiogenic marker FGF2.The major weaknesses of the study in the search for novel candidate markers are the relatively heterogeneous study population and the low number of patients included in the analysis. Many of the results presented herein are therefore only borderline significant (or some only showing a trend) and further validation in a larger NSCLC set, as done for miR-155, is a prerequisite for firmer conclusions. This is especially the case when comparing significant differences between tumor tissues  as there, in general, are larger differences between tumor tissues and normal controls. The fold changes are in average higher and the p-values stronger in Strengthening our results are strong and significant correlations between the microarray hybridization data and the same miRs validated by qPCR. Further, the altered miR expressions in tumor versus normal tissue are to a large degree consistent with previously published NSCLC miR studies Few studies have explored the impact of pathways related to miR gene targets in NSCLC. The interpretation of such analyses is not straight-forward as the results are highly dependent on which target genes are linked to the miRs and which miRs are connected to the different pathways. In addition miRs are often multifunctional and the same miR may target diverse genes in different pathways, and a gene may be targeted by several miRs. Although not statistically significant, the GSEA ranged angiogenesis as the most important pathway in our NSCLC samples. There are several novel reports connecting specific miRs to angiogenesis supporting our findings In a comprehensive murine study, Olson et al. identified specific miR expression signatures associated with steps in tumorigenesis and different hallmarks of cancer, including angiogenesis miR-126 has recently been reviewed as an important player in angiogenesis miR-155 is one of the miRs most consistently involved in neoplastic disease In this study, miR-21 is significantly up-regulated in tumor versus normal tissue , supportThe Olson study indicates miR-424 to be involved in angiogenesis as this miR was down-regulated after sunitinib treatment Another significant up-regulated marker in our material is miR-210 , which pmiR-182 is included in the angiogenesis pathway in the GSEA, partly because fibroblast growth factor receptor substrate 2 (FRS2) is one of its target genes. FRS2 is a major regulator of the fibroblast growth factor pathway that we and others have shown to be important in NSCLC progression, potentially by stimulating angiogenesis We observe the same trend for miR-106a, which has been shown to be up-regulated during hypoxia in breast and colon cancer cell lines As expected, many miRs known to be associated with angiogenesis were not significantly altered in our study. Cluster miR-17-92 , let-7, let-7f, miR-27b, miR-15b, miR-16, miR-92a, miR-99a, miR-130a, miR-214, miR-221, miR-222 and miR-296 as well as some of the miRs presented in the heat map , were noAngiogenic inhibitors in combination with chemotherapy are established as NSCLC treatment, but a further knowledge on biology, effectors mechanisms and prognostic and predictive markers are warranted. miRs are stable and potentially measurable in both tissue and serum. Several miRs are linked to angiogenesis and this study supports the assumption that a number of angiogenesis-related miRs are potentially important in NSCLC progression. We propose miR-21, miR-106a, miR-126, miR-155, miR-182, miR-210 and miR-424 to be among the most interesting candidates. Since miRs often are stage and tissue specific, large scale studies are warranted to further explore their prognostic and predictive impact. Besides the same miR often has multiple target genes and different functions. Therefore functional studies are highly needed to gain better insight into their biological functions.The study is approved by The National Data Inspection Board, The Regional Committee for Research Ethics and Biobank Board Collection of Tissue. Information and subsequent written consent from patients or next of kin was considered, but as this was a retrospective study with more than half of patients deceased, some more than ten years ago, The Regional Committee for Research Ethics specifically waived the need for consent.From a previously well-described large NSCLC cohort RNA was isolated from the collected core samples by using the Recover All™ Total Nucleic Acid Isolation Kit for FFPE Tissues , according to the manufacturer's instructions, with some minor adjustments. Xylene treatment was increased from 3 min to 8 min, and protease treatment was increased from 3 hrs to approximately 20 hrs. RNA quality and quantity was assessed by using the NanoDrop 1000 spectrophotometer  and further verified by an Agilent 2100 Bioanalyzer profile.Tm LNA Array power labelling kit  following the procedure described by the manufacturer. The Hy3™-labeled samples and a Hy5™-labelled reference RNA sample  were mixed pair-wise and hybridized to the miCURYTm LNA Array version 5th generation , which contains capture probes targeting all human miRNAs registered in the miRBASE version 14.0 at the Sanger Institute. The hybridization was performed according to the miRCURY™ LNA array manual using a Tecan HS4800 hybridization station . After hybridization the microarray slides were scanned and stored in an ozone free environment (ozone level below 2.0 ppb) in order to prevent potential bleaching of the fluorescent dyes. The miCURY™ LNA Array microarray slides were scanned using the Agilent G2565BA Microarray Scanner System  and the image analysis was carried out using the ImaGene 8.0 software . The quantified signals were background corrected with offset value 10 and normalized using the global Lowess  regression algorithm Total RNA (700 ng) from sample and reference was labelled with Hy3™ and Hy5™ fluorescent label using the miRCURYhttp://www.ncbi.nlm.nih.gov/geo/). The GEO accession number for the platform is GSE27705 and the study may be viewed at: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE27705.The microarray data were prepared according to minimum information about a microarray experiment (MIAME) recommendations One or several of the following criteria were used when selecting miRs for PCR validation; significant P-value, high fold change and/or angiogenesis-related miRs. Five samples from each group with sufficient tumor miR remaining after the array analysis were used in the PCR validation. Out of the 41 tested miRs, 28 miRs were detected in 7 or more samples and analysed. The stability of the miRNAs detected in all samples was tested by Exicon using the Normfinder software. miR-423-5p was one of the recommended reference miRNAs together with miR-150, miR-423-5p and miR-300. miR-300 was excluded due to very low levels detected. Further Normfinder found large variation in miR- 150, however miR-423-5p was found to be the most stable miRNA in the data set and consequently used as reference. All real-time qPCR experiments were performed by Exiqon, Vedbaek, Denmark. 10 ng RNA was reverse transcribed in 10 µl reactions using the miRCURY LNA™ Universal RT miR PCR, Polyadenylation and cDNA synthesis kit (Exiqon); each sample was processed in triplicates. cDNA was diluted 100× and 4 ul was used in 10 ul PCR reactions according to the protocol for miRCURY LNA™ Universal RT miR PCR; each miR was assayed once by qPCR in triplicate cDNA. The amplification was performed in a LightCycler® 480 Real-Time PCR System (Roche) in 384 well plates. LinRegPCR (version 11.5) software was used to determine the qPCR amplification efficiency. The average amplification efficiency was used to correct the Raw Cp values.http://www.mirdb.org)  database who has compiled gene lists from pathways curated in the PANTHER database.The impacts of the different pathways were evaluated by Gene Set Enrichment Analysis (GSEA) derived from Protein ANalysis THrough Evolutionary Relationship (PANTHER). The PANTHER classification system is a unique resource that classifies genes by their functions, using published scientific experimental evidence and evolutionary relationships to predict function even in the absence of direct experimental evidence. The gene set was taken from the MirDB (http://mirdb.org/cgi-bin/pathway.cgi. miRNA annotation is based on miRBase version 13 (http://www.mirbase.org/). GSEA analysis was carried out using the R statistical language (www.r-project.org) and the GSEA package for R available at http://www.broadinstitute.org/gsea/downloads.jsp.A full list is available at The data were analyzed using Linear Models and Empirical Bayes Methods for Assessing Differential Expression in Microarray experiments (LIMMA) The enrichment score (ES) reflects the degree to which a gene set is overrepresented at the top or bottom of a ranked list of genes. Normalized enrichment scores (NES) are used to compare analysis results across gene sets. GSEA accounts for differences in gene set size and in correlations between gene sets and the expression dataset Pearson's correlation coefficient, r, was used to describe the association between array data and results from qPCR. Pearson Chi-Square and Fisher Exact Test were used to correlate angiogenic markers with miR-155.Figure S1In situ hybridization (ISH) analysis of NSCLC representing strong and weak intensities for tumor cell miR-155 expression. Negative (scramble-miR) and positive (U6) controls from the same tissue area are shown. Strong miR-155 staining (A) with corresponding negative (C) and positive (E) controls to the left. Weak miR-155 staining (B) with corresponding negative (D) and positive (F) controls to the right. ISH positive signals (miR-155 and U6) stain blue, while nuclei stain red.(TIF)Click here for additional data file.Table S1The 128 significant -up and down-regulated  miRs are presented with FDR adjusted p-values and respective fold changes .(XLS)Click here for additional data file.Table S2Comparison between the array and the qPCR data in validation set. The differences in average expression levels between the sample groups in the array study are shown in columns 2–4 . The differences in average expression levels between the sample groups in the qPCR study are shown in columns 5–7 . Positive numbers >1 are shown in red ( = fold change >2), negative numbers <−1 ( = fold change >−2).(DOC)Click here for additional data file.


Improved detection methods for diagnosis of malignant pleural mesothelioma(MPM) are essential for early and reliable detection as well as treatment.Since recent data point to abnormal levels of microRNAs (miRNAs) in tumors,we hypothesized that a profile of deregulated miRNAs may be a marker of MPMand that the levels of specific miRNAs may be used for monitoring itsprogress.miRNAs isolated from fresh-frozen biopsies of MPM patients were tested forthe expression of 88 types of miRNA involved in cancerogenesis. Most of thetested miRNAs were downregulated in the malignant tissues compared with thenormal tissues. Of eight significantly downregulated, three miRNAs wereassayed in cancerous tissue and adjacent non-cancerous tissue sample pairscollected from 27 formalin-fixed, paraffin-embedded MPM tissues byquantitative RT-PCR. Among the miRNAs tested, only miR-126 significantlyremained downregulated in the malignant tissues. Furthermore, theperformance of the selected miR-126 as biomarker was evaluated in serumsamples of asbestos-exposed subjects and MPM patients and compared withcontrols. MiR-126 was not affected by asbestos exposure, whereas it wasfound strongly associated with VEGF serum levels. Levels of miR-126 inserum, and its levels in patients' serum in association with a specificmarker of MPM, SMRPs, correlate with subjects at high risk to developMPM.We propose miR-126, in association with SMRPs, as a marker for earlydetection of MPM. The identification of tumor biomarkers used alone or, inparticular, in combination could greatly facilitate the surveillanceprocedure for cohorts of subjects exposed to asbestos. Malignant pleural mesothelioma (MPM) is an aggressive tumor with poor prognosis,mostly linked to asbestos exposure MPM is characterized by a long latency period from the time of asbestos exposure toclinical diagnosis, suggesting that multiple somatic changes may be required for thetumorigenic conversion of normal mesothelial cells It is known that epigenetic mechanisms are involved in the regulation of microRNAs(miRNAs) Aberrant expression of miRNAs has been shown to contribute to the pathogenesis ofseveral human diseases In this study, the miRNA profile associated with the development of MPM was evaluatedby quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analysisof biopsies freshly collected from patients with MPM and from healthy subjects usedas controls. Certain deregulated miRNA species were selected and subjected tofurther analysis in a larger series of samples. Among the miRNAs analyzed, themiR-126 was found to be significantly downexpressed in malignant tissues. Next, wetested the applicability of the selected miR-126 as a circulating biomarker forearly detection of MPM and risk-disease prediction. Thus, the miR-126 levels wereevaluated in serum samples of asbestos-exposed subjects, defined as high-riskpopulation to develop the disease, and MPM patients, correlated with the level ofthe angiogenic factor VEGF and SMRPs, and compared with healthy subjects. Ourresults indicate that the miR-126 expression, in particular in combination withSMRPs, may be used as a marker to help diagnose of the neoplastic disease and couldgreatly facilitate the surveillance procedure for cohorts of subjects exposed toasbestos.To determine the miRNA species differently expressed in the MPM tissue comparedwith the normal mesothelial tissue, we used a customized miRNA PCR array with 88human miRNAs that are known to play a role in cancer. By comparing miRNAs fromfreshly collected MPM biopsies with pooled miRNAs from normal controls, a miRNA‘signature’ was obtained. Most of the miRNAs were downregulated inthe malignant tissue compared to the healthy control samples . The mosThree miRNA species most consistently downregulated, with fold change of >15,were selected for a larger-scale analysis using the paraffin-embedded tissuesections. We therefore analyzed the expression of miR-335, miR-126 and miR-32 inthe pairs of the cancerous and the adjacent non-malignant tissue. The ROC curveswere generated to analyze the diagnostic value of individual miRNA. When thenumber of samples was increased, miR-335 lost its significance to discriminatethe pathological tissue (MPM group) from the normal, control tissue (NM group),accuracy to 42% .Therefore, only miR-126, accuracy to 70% , and, to a lower extent, miR-32, accuracy to65% , weresignificantly downregulated in the MPM group compared to the NM group allowingto discriminate between cancerous and non-malignant tissues .The MPM group was then divided into two sub-groups according to the stage of thedisease. The MPM tumors staged S-Ia, S-Ib, or S-II without lymph nodes andmetastases involvement were included in the S1 group, while the MPM tumorsstaged S-III or S-IV with lymph nodes and metastases were included in the S2group. We then compared the expression of miR-335, miR-126 and miR-32 in thesetwo groups. Although without reaching statistical significance, all miRNAs weremore downregulated in the advanced S2 group .Recently, the expression profile of circulating miRNAs in the serum has beensuggested as a potential biomarker for cancer detection MiR-126, the most important marker in our previous screening (see above), wasevaluated in a cohort of asbestos-exposed subjects defined as high-risksubjects, in MPM patients and in healthy controls.p = 0.02). As shown in T = −3.5;sensitivity 60% and specificity 74%) and from MPM patients.MiR-126 was correlated with the serum levels of the angiogenic factor VEGF andthe SMRPs, a specific marker of MPM To evaluate whether a combination of individual markers may increase thepredictive value for early detection of MPM, SMRP levels, that can distinguishMPM patients from asbestos-exposed subjects and controls with a cut-off of 1 nM,have been found to correlate with serum miR-126 . The proThe prognosis of MPM patients is dismal despite current therapeutic modalities thatinclude surgery, chemotherapy, and radiation of the thoracic drainage site Focus has been on finding tumor markers that can be used in association withradiography for MPM detection Accumulating reports strongly indicate the potential diagnostic applications of miRNAin human cancers, also suggesting their possible use in therapeutic applicationscfTo identify a specific miRNA signature, we first analyzed human miRNAs with apotential role in malignant tissue freshly obtained from MPM patients, which wascompared with corresponding samples of normal human mesothelium. This approachrevealed differences in the expression profile of miRNAs in MPM samples and in thecontrols. We found that most miRNA species were expressed at lower levels in the MPMsamples compared to the controls cf. This reAn miRNA profile was previously identified by analyzing 17 biopsies freshly collectedfrom MM patients for 723 human and 76 viral miRNAs cfIn this study, the miRNA profile was determined using biopsies collected from MPMpatients before the diagnosis and, therefore, the enrolled patients did not receiveany adjuvant chemotherapy or radiation therapy that could affect expression of theindividual miRNAs. Also, the qRT-PCR array we used allowed quantification of miRNAexpression. Using this approach, we identified eight significantly downregulatedmiRNAs. Of these, three most consistently deregulated miRNAs were analyzed in 27cancerous and adjacent non-malignant tissue sample pairs, resulting in theidentification of miR-126 as promising markers that may be potentially utilized todistinguish cancerous and normal tissue. More specifically, we observed low levelsof miR-126 in MPM samples, with their expression independent of tumour stagingcf,3.The expression of miR-126 has been recently found to be low in human lung cancercells. More specifically, miR-126 has a binding site in the 3′-untranslatedregion (3′-UTR) of the VEGF-A mRNA, and its upregulation resulted in decreasedexpression of VEGF-A. These results suggest a tumor suppressor function of miR-126in the context of lung cancer cfcfcfOne of the major challenges in MM is the identification of biomarkers for earlydetection of the disease, which can be routinely measured in surrogates. Recently,circulating miRNAs have been shown as promising biomarkers for detection of humancancers In this study, we have identified miRNAs whose expression differs in the MPM tissuewhen compared to the corresponding healthy tissue. Of the various differentiallyexpressed miRNAs, miR-126 was found to be significantly downregulated in themalignant tissue. Further, expression of miR-126 can be easily evaluated in theserum, and its level in association with a specific marker of MPM, SMRPs, can beused to identify subjects with high risk to develop the disease. The identificationof tumor biomarkers used alone or, in particular, in combination could greatlyfacilitate the surveillance procedure for cohorts of subjects exposed to asbestos, arelatively common phenomenon in different areas of industrialized countries.All subjects filled a questionnaire including their informed consent. The studywas carried out according to the Helsinki Declaration and the samples wereprocessed under approval of the written consent statement by Ethical Committeeof the University Hospital of Marche, Italy.To obtain biopsy specimens, 22 subjects  who underwent thoracoscopy or thoracotomy for suspected MPM wereenrolled. The collected tissue was divided into two parts; one was immediatelysuspended in the RNALater solution  and stored at−80°C until RNA extraction. The other tissue portion was used forhistological examination by the Pathological Anatomy Unit of the HospitalUniversity of Ancona, Italy. According to the diagnosis, the individuals wereclassified as subjects with MPM (the MPM group) and as healthy subjects withnormal mesothelium (the NM group). The MPM group included tissue with clearsigns of the pathology (n = 10), while the NM groupincluded non-malignant tissue (n = 5). The exclusioncriteria were the presence or suspicion of any infectious disease and othermalignancies. Tumors were classified as epithelioid, sarcomatoid, biphasic, andthe tumor stage evaluated based on the recommendation by the InternationalMesothelioma Interest Group (IMIG) Formalin-fixed, paraffin-embedded (FFPE) tissue of the subjects affected by MPM(n = 27) was collected from the Archive of the PathologicalAnatomy Unit of the Hospital University of Ancona, Italy. The FFPE samples werecut into 5 µm sections and stored at room temperature until analysis. Theadjacent non-cancerous tissue was used as a control. The clinical data wereobtained retrospectively and included information on the gender, age, histology,neoadjuvant chemoradiation and therapy administration (before surgery), smokingstatus and the pathologic staging. The MPM sub-types were as follows: 23epithelioid, 1 sarcomatoid and 3 biphasic. The demographic and pathologicalcharacteristics of the subjects are summarized in f) are interpreted as thecumulative dose of asbestos fibers in the workplace of(ff/cm3)×yrs f of 28.8±50.4 (ff/cm3)×yrs. Smokers87/196 (44%), ex-smokers 31/196 (16%) and non-smokers 78/196(40%) were examined. Evidence of asbestos-related diseases  was found in 56/196 (29%) subjects.From November 2004 to April 2010, 196 subjects  with a history of asbestos exposure wereenrolled at the Institute of Occupational Medicine, Polytechnic Universityof Marche, Ancona, Italy. The participants were interviewed by trainedpersonnel and answered a detailed questionnaire on duration of asbestosexposure, smoking and occupational tasks. Each subject underwent lungfunction analysis, chest radiography, and high-resolution computedtomography. A ‘fiber-year’ exposure metric was calculated foreach subject, assigning to each person an arbitrary coefficient of‘inhalated fibers (ff)’ indicating the occupational hazard. The‘cumulative fibers’  diagnosed for MPM,were recruited, from November 2004 to January 2010, at the Oncology Clinicof the University Hospital of Ancona, Italy, and included smokers 18/44(41%), ex-smokers 7/44 (16%) and non-smokers 19/44(43%). Exclusion criteria were the presence or suspicion of anyinfectious disease, previous radical surgery, radiotherapy, as well aschemotherapy for MPM. Pathological diagnosis was performed on pleuralbiopsies obtained by thoracoscopy or thoracotomy. Tumors were classified asepithelial in 30, mixed in 8 and sarcomatoid in 6 patients, and the tumorstage was evaluated.The control group consisted of 50 healthy subjects  recruited from November 2004 to January 2010,and included smokers 27/50 (54%), ex-smokers 4/50 (8%) andnon-smokers 19/50 (38%). The subjects were undergoing screeningradiography for chemoprevention at the Pneumology Clinic of the UniversityHospital of Ancona, Italy. None of them had ever been occupationally exposedto asbestos as documented by their occupational histories, and theypresented with normal chest radiographs. Venous blood was collected fromeach subject at the time of clinical examination and serum prepared.2 qPCR-grade miRNA isolation kit, and the cDNA synthesized using theRT2 miRNA First Strand kit (SABiosciences) according to themanufacturer's instructions. The expression of 88 miRNA species involved inhuman cancer development  was assessed by qRT-PCR  using RT2SYBR Green qPCR Master Mix (SABiosciences).Total RNA was extracted from biopsies using Tri-Reagent  according to the manufacturer's instructions. MiRNAs were isolatedfrom total RNA by the RTTotal RNA from the FFPE tissue samples (10 µg) was obtained using theRecoverAll total nucleic acid isolation kit  accordingto the manufacturer's instructions. Expression of selected miRNA specieswas quantified by qRT-PCR  using the TaqMan MicroRNAAssay .Circulating RNA was isolated by adding to 250 µl of serum an equal volumeof Tri-Reagent BD , the phase lock gel (Eppendorf) was usedto improve RNA recovery. The miRNA isolation kit (SABiosciences) was used formiRNA purification. miRNAs were eluted in a final volume of 40 µl RNasefree water and 4 µl were reverse-transcribed to cDNA using individualTaqMan MicroRNA Assay and the expression quantified by qRT-PCR.To normalize the expression levels of target miRNA, the U6 small nuclear RNA wasused as a control (housekeeping).The level of SMRPs was assessed using a sandwich-type ELISA assay  according to the manufacture's instructions, andthe results are expressed in nmol/l. Briefly, 100 µl of standard andplasma samples (1∶100 dilution) were added to each well of a 96-wellmicrotitre plate coated with specific antibodies against SMRPs and incubated atroom temperature for 60 min. After washing, the plate was incubated with asecondary HRP-conjugated antibody. The detection process included the additionof 100 µl of the TMB substrate to each well and the absorbance was read at 405 nm using an ELISAplate reader . Concentrations of SMRPs wereextrapolated from the standard curve, and expressed in nM.Human VEGF ELISA kit  was used according to themanufacturer's instructions to assess levels of the cytokine in serumsamples. The results are expressed in pg/ml.T(CT of miRNA - CT of housekeeping) and high miRNAΔCT value corresponded to low miRNA expression. The foldchanges in relative miRNA expression were calculated using the equation2−Δ(ΔCt). MiRNA species that were not detected inany of samples or with a CT value >35 were excluded from thecomparison. Differences with p<0.05 were consideredstatistically significant.The results were expressed as mean±S.D. of ΔCT log values, and the resulting expressionmap was visualized with Treeview using the average-linkage clustering algorithms. The miRNA species with increasedexpression are indicated by red color, those with decreased expression are shownin green color. Yellow color indicates miRNAs whose expression was similar inthe MPM and NM groups. Black color indicates miRNAs that were not detected.The miRNA species with at least a two-fold expression change between groups wereconsidered differentially expressed. The cluster analysis was performed on thebasis of the ΔΔCStatistical significance of different expression between two groups wasdetermined by means of the t-test and paired t-test. Multiple comparisons weredetermined by Analysis of Variance (ANOVA) followed by the Post-hoc LSD test.Correlations were performed according to Pearson. Receiver operatingcharacteristics (ROC) curves were plotted to quantify the marker performance.The area under curve (AUC) indicates the average sensitivity of a marker overthe entire ROC curve. The robustness of the models was evaluated using bootstraptechniques. The best statistical cut-offs were calculated by minimizing thedistance between the point with sensitivity = 1 andspecificity = 1 and the intercept on the ROC curve.Multivariate logistic regression model was used to estimate the influence ofindependent variables such as asbestos-related diseases , duration of asbestos exposure, cumulative fibre doses, VEGF and SMRPlevels on the selected miRNA.The data were analyzed by the Statistical Package Social Sciences (version 15)software .


The role of high-dose therapy (HDT) followed by autologous stem cell transplantation (ASCT) in the treatment armamentarium of aggressive B- and T-cell non-Hodgkin lymphoma (NHL) is still a matter of debate. In the pre-Rituximab era, the PARMA study demonstrated the superiority of HDT/ASCT over conventional salvage chemotherapy in chemosensitive, relapsed patients. Subsequently, HDT/ASCT has become a standard approach for relapsed NHL. With the advent of Rituximab in the landscape of NHL, transplantation as part of first-line therapy has been challenged. However, no benefit in terms of disease-free or overall survival of HDT/ASCT over standard therapy was shown when Rituximab was added to both arms. Moreover, the superiority of HDT/ASCT over conventional salvage therapy in patients relapsing from first-line therapy including Rituximab was not confirmed. From these disappointing results, novel strategies, which can enhance the anti-lymphoma effect, at the same time reducing toxicity have been developed, with the aim of improving the outcome of HDT/ASCT in aggressive NHL. In T-cell lymphoma, few publications demonstrated that consolidation of complete remission with HDT/ASCT is safe and feasible. However, up to one-third of patients may never receive transplant, mostly due to progressive disease, and relapse still remains a major concern even after transplant. Aggressive lymphomas comprise a variety of lymphoid malignancies which can benefit in various phases of the diseases from high-dose therapy (HDT) followed by autologous stem cell transplant (ASCT). Despite data suggesting that prolonged event- event free survival can be achieved with SCT combined with HDC, there are problems that may limit the utility of this approach for a broad patient population. Moreover, the development of Rituximab has generated a sort of skepticism on the usefulness of HDT/ASCT, especially Mediterr J Hematol Infect Dis 2012; 4; Open Journal System in front-line therapy. Consequently, there is an urgent need for other effective and well-tolerated approaches that will eradicate the residual disease that may persist before ASCT, thus improving outcomes for patients with this life-threatening disease. In addition, approaches with better safety profiles would allow older patients to benefit from this therapeutic option.In this review, we will deeply discuss the data available about HDT followed by ASCT in aggressive lymphomas.On the first decade of study into autologous transplantation for the treatment of aggressive lymphoma, the focus was on the use of this approach to rescue patients after relapse or if the disease already progressed under standard chemotherapy. These encouraging results in relapsed or progressive lymphoma led to the testing of the technique as a primary therapy for the disease.Thirteen prospective multicenter randomized studies have tried to evaluate the impact of HDT with ASCT as part of the first-line treatment for patients with diffuse large B cell lymphoma (DLBCL) .1–12 In In order to assess the effects of such high-dose treatment on overall survival in patients with aggressive non-Hodgkin lymphoma, the Cochrane Hematological Malignancies Group performed in 2008 a meta-analysis that included data from 15 randomized controlled trials with a total of 3079 patients treated for aggressive NHL.Overall, with respect to the large population included in the meta-analysis and the attempts made to minimize bias; the Authors concluded that there is no evidence for a general benefit of HDT followed by ASCT for patients with aggressive NHL as first-line treatment based on the data available at the time of writing (2008). In fact, the improvements in relapse free survival and complete remission rates did not translate into an overall survival benefit in the respective groups. However, if HDT is employed for high risk patients, there may be a benefit.With the remarkable increase in the response rate when Rituximab (R) was added to conventional chemotherapy, for new studies comparing standard chemotherapy to HDT followed by SCT with the incorporation of R into both treatment concepts were required. Several phase II studies and phase III randomized trials investigated the role of HDT followed by ASCT for the initial treatment of DLBCL patients .14–20 TaVitolo et al.The GELA groupA case-control study by matching (1:1) patients treated with R with patients treated with identical chemotherapy  program not given R but submitted to ASCT was also performed in both the Italian and French studies. Regardless of the limitations intrinsic to retrospective analyses, these comparisons demonstrated a marked therapeutic advantage of chemo-immunotherapy over chemotherapy alone in both PFS and OS.The US/Canadian Intergroup trial (SWOG S9704)2). Patients with CR or PR continued treatment as initially randomized with 4 courses of RCHOP-14, 2 courses of R-megaCHOP or R-MAD  in the absence of response, in all cases followed by BEAM/ASCT. Again, 2-year-PFS was significantly higher for patients receiving HDT/ASCT as compared to R-(mega)CHOP-14 (p =0.0128), but again OS was not significantly different.The Italian lymphoma foundation (FIL)Both the SWOG and the FIL study randomized only patients with chemosensitive disease: patients without CR or PR after (R)-chemo were not eligible for transplantation. However, while the Italian study indicate that chemosensitive patients may significantly do better with HDT/ASCT with respect to further conventional therapy also when R is a part of frontline therapy, the results of the SWOG study are not conclusive because the comparison of 8 courses of R-CHOP-21 and 5 coursed of R-CHOP-21 followed by HDT/ASCT lacks the statistical power necessary to show significant differences.The German groupThe GELAIn conclusion, results of all these studies incorporating HDT/ASCT in the frontline therapy of aggressive B-cell lymphoma are contradictory and definitive conclusions may indeed not be drawn.The superiority of HDT and ASCT over conventional salvage chemotherapy was demonstrated by Philip et alRecently, the randomized CORAL study,Due to the results reported from the randomized CORAL study which are less favorable than those reported in non-randomized study,Initial studies in relapsed aggressive NHL incorporated total body irradiation (TBI) into the conditioning regimen approach as the mainstay of therapy for chemosensitive NHL. This approach was considered reasonable and effective at the time. Tissue dose limitations, however, prohibit the use of TBI in patients who have received prior consolidative or salvage radiation after initial chemotherapy. More importantly, a significant proportion of the patients treated with ASCT subsequently developed secondary myelodysplasia or acute leukemia. Most transplant centres and groups have moved away from TBI-conditioning approaches and have concentrated on chemotherapy-based regimens.42Chemotherapy-based regimens consist of 2–4 drug combinations including an alkylating agent given over 6–7 consecutive days. These combination treatments induce CRs in the 60–85% range while at 2–5 years after ASCT have resulted in 34–60% DFS and 24–46% OS rates. TRM have resulted in 3.8–17%.2 for 2 days). The median age was 43 and DLBCL was the common histological subtype. At a median of follow up of 16.4 months, 15 patients (23.4%) exhibited a relapse or progression, while 13 patients (20.3%) had died of disease. The estimated 3-year OS and PFS for all patients was 72.1 and 70%, respectively demonstrating the efficacy and tolerability of this conditioning regimen.Kim JG et al.2, 180 mg/m2 and 200 mg/m2 given on days -7 and -6) coupled with fixed doses of etoposide, cytarabine and melphalan (BeEAM regimen) for resistant/relapsed lymphoma patients. The basis for testing bendamustine in a novel conditioning regimen arose from preliminary in vitro data on lymphoma cell lines, demonstrating the higher efficacy bendamustine has in combination with etoposide, cytarabine and melphalan with respect to carmustine. 28 NHL patients were enrolled in the study, which also comprises 15 Hodgkin lymphoma patients. In total, 43 patients received a median number of 6x106CD34+cells/kg (range 2.4–15.5). All patients engrafted, with a median time to ANC>0.5x109/l of 10 days. All patients fully engrafted, with a median time to achieve ANC>0.5x109/L and PLT>20x109/l superimposable to those of other conventional conditioning regimens . The 100-day transplant-related mortality was 0%. After a median follow-up of 30 months (last update June 2012), 32/43 patients (74%) are in complete remission, whereas 11/43 relapsed and 3/43 died. Five out of 28 NHL patients (17%), as well as 6/15 (40%) HD patients relapsed. Disease type (NHL versus HD) and disease status at transplant (chemosensitive vs chemoresistant) significantly influenced disease-free survival . Remarkably, 4/43 (9%) patients achieved the first complete remission after receiving the high-dose therapy with ASCT. We concluded that the BEAM regimen is safe and effective for heavily pretreated lymphoma patients, with 74% of patients being alive and disease-free after a median follow-up of 30 months. Further studies are warranted to confirm these preliminary results. For this reason, we are currently running a new study testing Bendamustine at 200 mg/m2/day over 2 days in aggressive (DLBC or grade III follicular lymphoma) B-cell lymphoma.Our group2/min in sequential cohorts, in daily 3- or 2-dose schedules. Each dose immediately preceded busulfan or melphalan. 133 patients were enrolled . Primary refractory disease was present in 45% of NHL patients, and 50% of patients were PET-positive at transplant. The 2-dose schedule was better tolerated. Overall response and CR rate were 100% and 69% respectively in aggressive B-cell NHL, and 66% and 66% in T-cell NHL. After a median of follow up of 24 months, EFS and OS rates are 60 and 89% respectively (B-NHL), and 70% and 70% (T-NHL).Nieto Y et alIn parallel to novel chemotherapeutic agents, several investigators tested the safety and the efficacy of adding or incorporating radioimmunotherapy in HDT prior to ASCT.Radioimmunotherapy is a novel type of immunotherapy that uses a linker to combine a monoclonal antibody with a certain specificity linked with a radioimmunoconjugate. The currently available agents used in lymphoma target the CD20 molecule. Two radiolabeled antibodies, iodine-131 tositumomab and yttrium-90 ibritumomab, have been approved by the US Food and Drug Administration (FDA) to treat relapsed lymphoma.Radiation therapy has been a component of the conditioning regimens used to treat lymphoma in the past. Because lymphomas are highly sensitive to radiation, radioimmunotherapy (RIT) has been used with great success in consolidation therapy and, as a result, either as a single agent or as augmentation of HDT, as part of a conditioning regimen for ASCT. The flexibility of including RIT as part of conditioning therapy also allows it to be combined with RIC to reduce the toxic effects of HDC. In fact, RIT delivers targeted radiation to lymphoma sites protecting other tissues; therefore, it limits toxicity and allows the use of ASCT in older patients or in patients with comorbidities and decreased organ function. This treatment option replaces any concomitant loss of chemotherapy efficacy with a gain in RIT efficacy. The data so far suggest that the use of RIT in the autologous setting can improve clinical outcome with no added toxicity in these patients, whereas similar positive findings have been reported in preliminary studies of RIT combined with RIC and alloSCT in high-risk patients.Both antibodies have been used in phase I and phase II clinical trials, in conventional standard dose or escalated high dose, to increase the therapeutic effect of high-dose therapy or to substitute TBI, with the aim of reducing relapse rate without adding toxicity to the conditioning regimens. Table 3The Blood and Marrow Transplant Clinical Trials Network (BMT CTN) conducted a prospective multicenter randomized phase III trial (0401)Despite all the experience and information gathered on this disease there remains no regimen that has demonstrated superiority. Studies published in the Rituximab era appear to show less toxicity than the pioneering works of the 90’s, but no significant improvements in OS rates was demonstrated for patients submitted to HDT/ASCT.While salvage with R-chemotherapy followed by HDT/ASCT remains a viable option, new strategies have to be taken in account for the remaining patients with relapsed/refractory aggressive lymphomas.For the time being, allogeneic SCT and, very recently, T-replete haploidentical SCTPeripheral T-Cell lymphoma (PTCL) and natural killer/T-cell lymphoma constitute a rare and very heterogeneous group of NHL. In Western countries, they account for 10% to 15% of all adult lymphomas. With the exception of anaplastic large-cell lymphoma (ALCL) positive for anaplastic lymphoma-kinase (ALK), PTCL carries a poor prognosis with low OS and DFS with conventional chemotherapy.The most frequent T-Cell lymphoma is represented by PTCL not otherwise specified, ALCL and by angioimmunoblastic T-cell lymphoma (AITL).Five prospective studies incorporating HDT/ASCT as part of first-line therapy have been reported. Corradini et al.Reimer et. alThe Nordic Lymphoma Group (NLG)The Gel-Tamo Study Group investigator published their experience in 26 patients with PTCL, excluding ALK-positive ALCLMercadal et al.These publications  suggest High dose therapy followed by autologous stem cell transplantation (HDT/ASCT) together with allogeneic and haploidentical stem cell transplantation have still clear roles in the treatment armamentarium of an highly heterogeneous disease known as aggressive lymphomas.Regarding B-cell lymphomas, the recent incorporation of Rituximab into virtually all first-line therapies of all B-cell lymphomas resulted in major improvements of conventional therapy. Even if a consensus is a missing due to the fact that results of the studies incorporating HDT/ASCT in the frontline therapy of aggressive B-cell lymphoma are contradictory, a significant proportion of high risk (high aaIPI) may still benefit from HDT/ASCT, at least in terms of a longer disease-free survival.On the other hand, at the present time, patients with relapsed or refractory B-cell lymphoma are more difficult to salvage than in the pre-rituximab era. While salvage with R-chemotherapy followed by HDT/ASCT remains a viable option in the Rituximab era, it is surely not the best. Results from clinical trials are disappointing for relapsed patients, and thus novel strategies, which can enhance the anti-lymphoma effect, at the same time reducing toxicity need to be exploited with the aim of improving the outcome of HDT/ASCT in aggressive NHL. Studies testing novel high-dose strategies prior to ASCT and studies comparing the efficacy of alloSCT and HDT/ASCT are warranted, and some of them are currently on their way.For patients with T-cell lymphoma, no major progress has been made over the last decade and phase III studies comparing chemotherapy to novel agents or combining them are still ongoing. However, no magic bullet such as Rituximab for B-cell lymphoma seems to stand in the landscape of T-cell lymphomas. In this regard, even if the results of some studies support the use of HDT/ASCT for patients with T-cell lymphoma, showing that a small proportion of patients with T-cell lymphoma may survive several years without disease after HDT/ASCT, we think that allogeneic SCT will play an increasing role for all patients with T-cell lymphoma, including high-risk patients needing first-line therapy.


Rabies virus (RABV) and European bat lyssavirus 1 and 2 (EBLV-1 and -2) RNA samples, with systems available in their laboratory.Rabies is a lethal and notifiable zoonotic disease for which diagnostics have to meet the highest standards. In recent years, an evolution was especially seen in molecular diagnostics with a wide variety of different detection methods published. Therefore, a first international ring trial specifically designed on the use of reverse transcription polymerase chain reaction (RT-PCR) for detection of lyssavirus genomic RNA was organized. The trial focussed on assessment and comparison of the performance of conventional and real-time assays. In total, 16 European laboratories participated. All participants were asked to investigate a panel of defined lyssavirus RNAs, consisting of The ring trial allowed the important conclusion that conventional RT-PCR assays were really robust assays tested with a high concordance between different laboratories and assays. The real-time RT-PCR system by Wakeley et al. (2005) in combination with an intercalating dye, and the combined version by Hoffmann and co-workers (2010) showed good sensitivity for the detection of all RABV samples included in this test panel. Furthermore, all used EBLV-specific assays, real-time RT-PCRs as well as conventional RT-PCR systems, were shown to be suitable for a reliable detection of EBLVs. It has to be mentioned that differences were seen in the performance between both the individual RT-PCR systems and the laboratories. Laboratories which used more than one molecular assay for testing the sample panel always concluded a correct sample result.Due to the markedly high genetic diversity of lyssaviruses, the application of different assays in diagnostics is needed to achieve a maximum of diagnostic accuracy. To improve the knowledge about the diagnostic performance proficiency testing at an international level is recommended before using lyssavirus molecular diagnostics e.g. for confirmatory testing. Rhabdoviridae family Rabies as a notifiable zoonotic disease is an acute, progressive and incurable viral encephalitis which is clinically characterized by central nervous disorders that ultimately lead to death. The disease is caused by different Lyssavirus species of the Whilst ante-mortem testing is only recommended by the World Health Organization (WHO) for rabies suspect human patients, definite rabies diagnosis in both human and animal samples relies on post-mortem laboratory findings. The widely accepted (post-mortem) “gold standard” method of the WHO and the World Organisation for Animal Health (OIE) is the detection of lyssavirus antigen by the fluorescent antibody test (FAT) With the advance of molecular techniques, reverse transcription polymerase chain reaction (RT-PCR) has been increasingly used for amplification of lyssavirus RNA from sample materials. Nowadays, numerous assays are available; for review see Dacheux et al. (2010) © assays detects and differentiates RABV from EBLV-1 and EBLV-2. Generic primers and species-specific probes were combined within one reaction For generic pan-lyssavirus approaches the hemi-nested assay described by Heaton et al. (1997) Newly available chemistries, reagents and procedures have improved and standardised the detection techniques leading to higher sensitivities and specificities. Therefore, validated RT-PCR-based tests were proposed as alternative, confirmatory tests also for rabies, and were suggested to be included in the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals Previous ring trials focussed mainly on classical methods like FAT and virus isolation or included only a very limited panel of brain tissues for the RT-PCR analysis 3) solution  in RNase free water) A panel of 28 lyssavirus samples from the virus archive of the Friedrich-Loeffler-Institut (FLI) was selected for the trial. Either original brain material or tissue culture supernatant after infection of MNA cells was used . All isoq values for real-time PCRs, respectively, had to be reported using an Excel-spread sheet. Each test was designated to the respective laboratory (designated as A-P), and if two or more methods were performed an additional number  was added. Furthermore, additional information on the established RT-PCR assays e.g. published or unpublished (in-house) method, RT-PCR kits used and modifications of protocols were also requested and recorded.All participants were asked to investigate the complete set of 30 blinded samples in duplicate with the diagnostic PCR assays used in their own laboratory. If possible, differentiation of the most common species  was requested and results, e.g. positive/negative for conventional and CIn summary, nine different published and five unpublished (in-house) assays mostly targeting the nucleoprotein gene were used in the frame of this ring trial, including real-time RT-PCR 14 labs) as well as conventional RT-PCR (5 labs) techniques , and 4.  labs as q values  a combination of R13 and R14 assays (in single wells or as duplex assay) were able to detect all rabies samples very robustly. However, sample L-29 was not detected by any of the applied R13 assays and samples L-05, L-12 and L-17 were unreliably detected. Samples L-09 and L-15 from Nigeria (clade Africa 2) were not detected by the R14 system (described previously by Lyssavirus species.The results from laboratories O (R13/14) and P2 (R14) for sample L-24 provide some indication for cross-reactivity of these rabies-specific methods with EBLV-2. Although this is not a diagnostic disadvantage, as the assay detected a positive lyssavirus sample correctly, it highlights the issues with the use of hydrolysis probes to differentiate q values  compared to laboratory A (A1). Also, the variation of the Cq values for individual samples between different laboratories using a lab-version of the same assay was remarkably broad in several cases.Varying the R14 single assay with different PCR chemistries, laboratories E and J produced identical seven false-negative results. Using this assay with the same RT-PCR kit and the same PCR cycler model but slightly different temperature profiles, laboratory P (P2) yielded a better performance with lower CThe assay published by Orlowska et al. (2008) q values for the recognition of EBLV-1.In general, EBLV-1 and -2 were recognized correctly by the appropriate assays . Only onThe performance of five conventional RT-PCR methods for RABV-specific detection or a broad range application is shown in Laboratory I (I7) recognized all isolates except the European sample L-17 with a two-step version of this assay. Lab-versions of the system by Echevarría and co-workers (2001) In general, usage of different tests in parallel led to correct or at least questionable results which would require further investigations . There aDuring the recent validation of a fluorogenic probe-based real-time RT-PCR for RABV, it had become evident, that a single assay was not sufficient to detect all tested RABV strains due to a high degree of genetic diversity The results described and discussed here revealed a relatively high degree of divergence among the participating laboratories. Partially, this can be explained by the diagnostic range of the applied assays and has been highlighted in In previous European ring trials for rabies routine diagnosis Ring trials as the one described here trigger diagnostic laboratories to start intensive investigations on the diagnostic quality, so that an overall improvement can be made.A first problem that may be associated with comparing the performance of RT-PCR assays is the stability of the RNA during both, transport and testing. In our case, RNA degradation is unlikely as the RNA was preserved in a special storage buffer and shipment was done on dry ice. Furthermore, a freeze-thaw control was used to confirm RNA stability. A similar approach proved to be successful for a recent European classical swine fever virus (CSFV) ring trial As a main conclusion the R13/14 RABV-specific real-time RT-PCR system In North America, TaqMan® PCR assays for the detection of RABV were either comparable, or they had a considerably reduced detection limit compared to hemi-nested PCR One argument against PCR diagnostics in the field of rabies, but also in general, is the risk of contamination leading to false-positive results. However, carry-over contamination from positive controls can be prevented efficiently by strict quality control procedures in place, such as using artificial positive controls Most conventional RT-PCR methods performed satisfactorily for RABV detection and in general were less error-prone; nearly all samples were recognized, than the real-time PCR approaches. Also one conventional RT-PCR assay generally failed to detect a certain isolate (L-15) presumably according to mismatches in the primer binding region due to viral diversity. Furthermore, there were fewer inter-laboratory variations when individual versions of the same assay were used. One possible explanation could be that conventional RT-PCRs are well established in many rabies diagnostic laboratories, whereas the implementation of real-time RT-PCRs is an on-going process. This is a very important point, as we would predict a similar situation with the real-time assays as they have become more embedded and routinely applied. While real-time RT-PCR assays could be used for rapid rabies diagnosis, conventional RT-PCR methods will remain valuable since sequence information can be obtained for subsequent phylogenetic analysis. Furthermore, all applied EBLV-specific investigations, real-time as well as conventional systems were suitable for a reliable EBLV diagnosis, although additional studies are required, since only single samples of each virus has been included into the study panel.q values in the high twenties, indicating a low amount of viral RNA. Thus by diluting further, some assays reached their diagnostic limits in terms of sensitivity. However, one has to keep in mind that false-negative results would be a major problem in animal rabies diagnosis where a human exposure occurred because this can cause human fatality. Thus, parallel testing is again recommended in this case.Based on molecular techniques using any kind of PCR diagnostics, 29 inconclusive or false negative results occurred resulting in an overall sensitivity of 93% (70.0–100%) for RT-PCR. Although this may be acceptable, in total, only four of the 16 laboratories submitted 100% concordant results for RABV diagnosis. This appears to be a very low proportion, considering that during rabies diagnostic ring trials 90.5% and 80.5% of participating laboratories produced satisfactory results with no false negative results in 2009 and one false negative result in 2010, respectively The overall analysis of the ring trial showed that RT-PCR could be a very reliable diagnostic tool if assays with the broadest diagnostic range are used and quality standards are met at each level. Then, RT-PCR methods are probably suitable to be used in a qualified and trained laboratory as a second diagnostic line in parallel to traditional methods like FAT, mouse inoculation test (MIT) and RTCIT. For this purpose, a further harmonisation and standardisation of the individual methods e.g. by the use of commercial RT-PCR kits as seen in the case of CSFV detection Here, we used a very broad and complex RNA panel to test the assays used as much as possible. Nevertheless, this ring trial was not intended to discredit any methodology. However, if a laboratory involved in rabies control uses a certain established protocol that solely recognizes the prevailing RABV variants, problems can occur if a case of imported rabies occurs. In this case, the molecular diagnosis needs to be as broad as possible. This is particularly important if laboratories act as national reference laboratories and are confronted with human rabies diagnosis. In this case, the use of broad spectrum lyssavirus assays may be more suitable, as no prior epidemiological information is necessary. These reference laboratories should particularly be interested in the realisation of further ring trials.At the moment, neither the WHO nor the OIE have approved molecular techniques for rabies. However, validated PCR-based investigations are proposed to be included in the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals Table S1Published conventional PCRs for lyssavirus detection and Published real-time PCR assays for lyssavirus detection.(DOC)Click here for additional data file.Table S2Participants of the lyssavirus ring trial and Overview of applied RT-PCR techniques and chemistries.(DOC)Click here for additional data file.Table S3Mean quantification cycle (Cq) values of a sample set testing before and after a freeze-thaw cycle (Freeze-thaw control).(DOC)Click here for additional data file.Table S4Overview of the overall results of laboratories which performed more than one assay.(DOC)Click here for additional data file.


Colorectal cancer affects over one million people worldwide annually, with the liver being the most common site of metastatic spread. Adequate resection of hepatic metastases is the only chance for a cure in a subset of patients, and five-year survival increases to 35% with complete resection. Traditionally, computed tomographic imaging (CT) was utilized for staging and to evaluate metastases in the liver. Recently, the introduction of hepatobiliary contrast-enhanced magnetic resonance imaging (MRI) agents including gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid  has proved to be a sensitive method for detection of hepatic metastases. Accurate detection of liver metastases is critical for staging of colorectal cancer as well as preoperative planning. Colorectal cancer is one of the most common cancers worldwide with nearly one million people diagnosed each year. The liver is the most common site of distant metastases from colorectal cancer. Up to 70% of all patients with colorectal cancer will develop hepatic metastases at some point in their lifetime, and one-third of these will have metastases confined only to the liver , 2. In metastases from colorectal adenocarcinoma, locoregional therapies are available including radiofrequency ablation and resection, which provide a survival benefit for patients with limited disease. Hepatectomy for liver metastases from colorectal cancer is the gold standard of treatment and provides the only chance for cure. Complete resection of all disease has been associated with a five-year survival ranging between 22% and 58% . However Historically, computed tomography (CT) has been used to stage and evaluate the liver in patients with colorectal adenocarcinoma. However, the introduction of magnetic resonance imaging (MRI) has proven to be a highly effective way to evaluate liver parenchyma –12. In 1Early publications evaluating the use of gadolinium-based contrast agents showed an advantage of MRI over CT . RecentlMangafodipir trisodium (MT) was the first specific hepatobiliary contrast agent. It was introduced in 1997 and approved as “an adjunct to MRI in patients to enhance the T1w images used in the detection, localization, characterization, and evaluation of lesions in the liver” .  MT isGadobenate dimeglumine  was approved by the FDA in 2004 for use as an MRI contrast agent. Gd-BOPTA acts as both an extracellular agent as well as a hepatobiliary contrast agent. While it is approved for hepatobiliary imaging in Europe, it is used off-label in the United States. 3% to 5% is taken up by hepatocytes and excreted into the biliary system which allows for its hepatobiliary specificity , 19. In 2008, gadoxetic acid  was FDA approved in the United States for the detection and characterization of liver lesions. It had previously been approved in Europe in 2004 as Primovist. Eovist is a gadolinium-based contrast agent with approximately 50% uptake into hepatocytes and subsequent biliary excretion . After aGd-EOB-DTPA's elimination half-life in healthy patients is roughly 55 to 57 minutes. Its elimination pathway is unique compared to other gadolinium agents. It is eliminated equally (50%) through the renal and hepatobiliary systems. In patients with one impaired pathway, the other elimination pathway will remove a larger percentage of the dose. For this reason, patients with renal or hepatic impairment do not require dose adjustment . It doesThe use of dynamic contrast-enhanced MRI with gadolinium-based contrast agents for the characterization of benign and malignant liver lesions has been well described in a growing body of literature , 31. LivGd-EOB-DTPA has unusual pharmacokinetics in that it is the only gadolinium agent which is taken up in equal amounts by the extracellular space and functioning hepatocytes, demonstrating a biphasic action. The dynamic phase of gadoxetic acid occurs during the first 2 minutes after injection. During this phase, normal hepatocytes as well as lesions containing hepatocytes exhibit contrast enhancement due to hepatocellular uptake. The enhancement pattern depends on vascular supply, the presence of functioning hepatocytes, and characteristics of the biliary structures within the lesion. The normal hepatocytes then slowly excrete the contrast agent into the biliary tree, which is referred to as the “hepatobiliary phase.” For gadoxetic acid, the duration of this phase is 20–120 minutes where hepatocytes have excreted the contrast into the bile and have reached a level optimal for interpreting images of both the biliary system and hepatic lesions , 27, 32.Metastatic lesions are typically present on gadolinium-based contrast-enhanced imaging with peripheral rim enhancement and lack of central enhancement in the dynamic phase when central tumor necrosis is present. During the hepatobiliary phase, contrast uptake by hepatocytes provides contrast between the liver parenchyma and metastatic disease causing metastases to appear hypointense. In addition, during the hepatobiliary phase, rim enhancement  and a “tInitial studies using gadolinium-based contrast agents showed advantages of MR over CT, but with some mixed results . Since tIn 2004, Kim et al. demonstrated improved tumor to liver contrast using gadobenate dimeglumine in the hepatobiliary phase, leading to detection of more metastases compared to dynamic imaging alone . In theiP < 0.001) difference in the correct detection with Gd-EOB-DTPA. It also showed a 7% increase in correct lesion classification with Gd-EOB-DTPA as compared to precontrast MRI [The European EOB Study Group  looked at 302 lesions in 13 patients with biopsy or intraoperative ultrasound proven focal liver lesions. 81 of these patients had metastases from a colorectal tumor primary. T1 and T2 phase MRIs, pre- and post-Gd-EOB-DTPA, were performed and evaluated in a blinded fashion by three radiologists. In 21 of the 129 patients, results differed between pre- and postcontrast MRI, and 19 of these were correct in the Gd-EOB-DTPA group, resulting in a significant  showed that the percentage of lesions that were correctly classified as malignant or benign was 2–15% greater for blinded readers comparing MR images with Gd-EOB-DTPA with helical CT images of the same patients, with an increase in false positive lesions identified using CT. False positive results in liver imaging for metastases from colorectal cancer can impact surgical planning, delay excision of the primary tumor, and result in unnecessary surgery . In 2006, Halavaara et al. showed superiority of Gd-EOB-DPTA compared to CT and diffusion weighted MR. They found increased lesion identification (95% versus 89%), sensitivity (95 versus 92%), and specificity (94 versus 90%) with MRI compared to CT . This waStudies by Ichikawa et al. (2010) have demonstrated superiority of gadoxetic-acid-enhanced MR over unenhanced MR and triphasic contrast-enhanced spiral CT for detection of metastases with regard to sensitivity. This was true for lesions <20 mm in diameter; however, the lesions <20 mm were not broken down by lesion type .In the same year, Shimada et al. looked at 45 patients undergoing abdominal MRI. A total of 51 hepatic metastases were examined by two independent observers. 7 of these lesions were seen on Gd-EOB-DTPA but were missed on DWI by both observers. 2 metastases very close in proximity to hepatic vessels were difficult to detect on Gd-EOB-DTPA but were seen clearly on DWI. In this study, Gd-EOB-DTPA-enhanced MRI showed statistically significant higher accuracy in detection of small lesions (<2 cm) than DWI. However, only slightly more than half of these metastases were confirmed histologically; the remainder were considered to be metastases on the basis of tumor growth on follow-up radiologic examinations .P = 0.002). There was also a significant difference in detection (29% and 71% P = 0.013) for lesions less than 1 cm in diameter. This study is very limited by the fact that it did not differentiate between benign and malignant lesions [P < 0.001). On subset analysis, 25 small lesions less than 1 cm in diameter were detected only with EOB-MRI [Two studies have compared Gd-EOB-DTPA-enhanced MRI with PET/CT. Donati et al. (2010) looked at 85 liver lesions in 29 patients. 45 of these were metastases from a colorectal primary. The metastases were not divided out by primary cancer type . When lo lesions . Followi EOB-MRI . This diLöwenthal et al. (2011) demonstrated the superiority of Gd-EOB-DTA-enhanced MR in the hepatobiliary phase to MR-DWI and MR for the detection of focal liver lesions when looking at 332 lesions . However, unlike Hammerstingl et al., this study did note that sensitivity for lesions <1 cm was higher for MR-DWI than for MR hepatobiliary phase images (0.98 versus 0.92) .Chung et al. (2011) looked at a series of 47 patients with a total of 78 confirmed colorectal metastases comparing DWI and Gd-EOB-DTPA-enhanced MRI. In this study, regardless of lesion size (greater or less than 2 cm in diameter), significantly more lesions were detected when looking at both DWI and Gd-EOB-DTPA-enhanced images than with DWI imaging alone . All lesMuhi et al. (2011) broadened the comparison and looked at the diagnostic accuracy of contrast-enhanced CT (CE-CT), contrast-enhanced ultrasound (CE-US), and superparamagnetic iron oxide-enhanced MRI (SPIO-MRI) for detecting colorectal hepatic metastases. 112 metastases in 46 patients were evaluated. For all lesions combined, sensitivity and area under the receiver operating characteristic curve of Gd-EOB-DTPA-enhanced MRI were significantly greater (95%) than CE-CT (63%) and CE-US (73%). For lesions less than 1 cm in diameter, sensitivity of Gd-EOB-DTPA-enhanced MRI was significantly greater than for CE-CT and CE-US. However, they did not find a significant difference in positive predictive value between any of the imaging modalities .P = 0.001) [Most recently, Chen et al. performed a meta-analysis of 1900 lesions from 13 studies showing the sensitivity of Gd-EOB-DTPA-enhanced MRI for detection of liver metastases to be 93% and specificity 95% with statistically significant decreased sensitivities with lesions less than 10 mm in size (= 0.001) . P < 0.001). There was not a significant difference in lesions >1 cm in diameter between MDCT and Gd-EOB-DPTA enhanced MRI (97% and 100% resp.) [The issue of imaging patients following neo-adjuvant chemotherapy who have developed hepatic steatosis was evaluated by Berger-Kulemann et al. (2012). In this study, 68 metastases were evaluated with triphasic MDCT and Gd-EOB-DTPA-enhanced MRI. All patients underwent surgical resection of liver metastases after evaluation. For lesions <1 cm diameter, MDCT detected only 41.9% while MRI detected 93% of metastases (% resp.) .While there have been multiple studies looking at the sensitivities and specificities of Gd-EOB-DPTA contrast enhanced MRI compared to other imaging techniques, economic considerations have become an increasingly important aspect of patient care in the United States. Zech et al. performed a cost analysis comparing MRI with Eovist, extracellular enhanced MRI, and three-phase MDCT as the initial evaluation of patients with metachronous colorectal liver metastases in Germany, Italy, and Sweden. It demonstrated that MRI with Eovist required fewer additional imaging studies (8.6%) than extracelluar-enhanced MRI (18.5%) and MDCT (23.5%). While MRI with Eovist has the highest initial imaging cost of the three modalities studied, it was in fact cost saving when reimaging, and the cost of modified and unnecessary surgical procedures was factored into the equation .The ideal preoperative imaging study would provide diagnostic information which is highly sensitive and has a low rate of false positives. The studies described above have shown that hepatobiliary phase imaging with gadoxetic acid is safe and offers increased sensitivity in the detection of metastases due to the superior liver-lesion contrast generated by the avid uptake of gadolinium into the background of liver parenchyma. In addition, it has a lower rate of false positives than helical CT scan. These are important aspects of imaging in preoperative planning for resection of metastases from colorectal cancer. Accurately mapping the location and number of metastases from colorectal cancer is crucial for the surgeon's preoperative planning process. Not only can it dramatically alter an operation from a minor wedge resection to a much larger anatomic procedure, but also it allows the surgeon to counsel the patient more accurately regarding the procedure they will require to excise the metastases. This is supported by the studies discussed previously where operative plans changed after reviewing Gd-EOB-DPTA enhanced MRI imaging . In addi