Genomic microarray analysis has revealed gene dosage changes adjacent to duplicons, but the high degree of similarity between duplicon sequences has confounded unequivocal assignment of chromosome breakpoints within these intervals. In this study, we localize rearrangements within duplicon-enriched regions of Angelman/Prader-Willi (AS/PWS) syndrome chromosomal deletions with fluorescence in situ hybridization (FISH).Segmental duplicons (SDs) predispose to an increased frequency of chromosomal rearrangements. These rearrangements can cause a diverse range of phenotypes due to haploinsufficiency, in Breakage intervals in AS deletions were localized recursively with short, coordinate-defined, single copy (SC) and low copy (LC) genomic FISH probes. These probes were initially coincident with duplicons and regions of previously reported breakage in AS/PWS. Subsequently, probes developed from adjacent genomic intervals more precisely delineated deletion breakage intervals involving genes, pseudogenes and duplicons in 15q11.2q13. The observed variability in the deletion boundaries within previously described Class I and Class II deletion AS samples is related to the local genomic architecture in this chromosomal region.Chromosome 15 abnormalities associated with SDs were precisely delineated at a resolution equivalent to genomic Southern analysis. This context-dependent approach can define the boundaries of chromosome rearrangements for other genomic disorders associated with SDs. These boundaries were approximated from both recombinant BAC sequences ) which is distal of BP3, and detects larger deletions [MAGEL2 [MIM 605283]).After bioinformatic analysis, 40 SC and LC intervals were marked for potential development of FISH probe reagents Figure . Nine ofeletions . SC probFISH studies with SC and LC probes validated the bioinformatic analysis. These probes can delineate boundaries of a chromosomal rearrangement in a single hybridization for certain breakpoints, however improved chromosome resolution was achieved through recursive hybridization using a series of probes. LC probes that detect fewer duplicon targets than anticipated from the genomic architecture; represent a deletion of one or more paralogs. Subsequent FISH analysis with adjacent SC probes from BP1 or BP3 can then determine which of the LC probe targets has been deleted. The process can be expedited by co-hybridizing the initial LC probe with a differentially-labeled SC probe adjacent to one of the duplicons. Figure Prior to undertaking this study, breakage intervals at the ends of the deletions in these AS cell lines were not precisely known, and the rearranged genes and SD features coincident with deletion boundaries had not been characterized.Variability in both the proximal and distal breakage intervals were observed in the three Class I AS cell lines Figure  such thaThe deletion breakage interval in BP3 was found to vary among different AS cell lines, with an overall distribution similar to those observed for Class I deletions Figure . At the We demonstrate that chromosome 15q11.2q13 deletions can be delineated using combinations of single and low-copy, sequence-defined FISH probes. The LC probes detect SDs, which are prone to rearrangement. Our analysis of these rearrangements distinguishes genes and pseudogenes at the boundaries of deletions that are either deleted or disrupted in AS.HERC2-containing SDs ), which do not overlap any duplicons, were deleted in all Class I cell lines. Dominant mutations in NIPA1 cause hereditary spastic paraplegia [Contextual mapping of 15q11.2q13 genomic rearrangements bracketed by SDs can distinguish genes that are disrupted from those that are have been demonstrated to be deleted using methods that quantify copy number, i.e. array CGH, qPCR, or MLPA. d Figure  and a pas Figure . TUBGCP5ntrosome ,29. CYFIith FMRP , which iith FMRP . NIPA2 [1 MIM 6062 was delGOLGA8DP  and GOLGA6L1 , are embedded within SDs. Both of these genes are likely to be disrupted or deleted in WJK67 and WJK70, but are intact in WJK36  and the GOLGA8E gene. Both GOLGA8E and HERC2P in BP2 are likely to be disrupted  was deleted in all cell lines [APBA2, which is distal to the telomeric HERC2 cluster in BP3, was intact. Large AS deletions that include APBA2 in 15q13.1 have been reported [Breakage intervals delineated in WJK67 and WJK35 overlap 2 Figure , 4D-IV. udogenes . Disruptnscripts . As expell lines ,35 and Areported  and a smreported .Sequence-defined SC and LC FISH probes will be generally useful to delineate genomic disorders bracketed by SDs that juxtapose during chromosome rearrangement. In AS/PWS, SMS, and DiGeorge syndromes, the distribution of SDs enables LC probes to be designed that simultaneously interrogate sequence copy number at both ends of the chromosomal deletion. However, as seen in 15q11.2q13, some SDs may contain remote paralogous targets that do not participate in the rearrangement. These intervals can still be useful as LC probes, with the remote paralogs serving as hybridization controls in metaphase FISH.Recently, recurrent microdeletions have been described in chromosomes 16p11.2p12.2 (chr16: 21.3-29.5 Mb) ,37, 1q21At the chromosome 1q21.1 and 3q29 loci [Interphase SC and LC FISH analysis may be feasible, if multiple, closely-spaced duplicon-related signals from an LC probe originate from the same homolog. We have not yet tested this possibility, given the highly decondensed state of interphase chromatin and the limited knowledge about spatial organization of homologous SDs during interphase.Breakage intervals of chromosomal rearrangements associated with SDs have been refined with several array CGH platforms -41. BAC Therefore, microarray designs generally avoid or provide low probe coverage in intervals that have genomic architectures such as SDs that confound the interpretation of copy number differences ,44. In rThe AS breakage intervals were also compared to the corresponding probe coverage by the Affymetrix SNP 6.0 microarray. Generally, there is a paucity of SNP probes within SDs. The breakage intervals delineated by SC and LC probes in the present study are not covered by any probes on the SNP array. However, several divergent SDs within BP1, BP2, and BP3 contained 3-10 probes targeting SNPs within duplicons. These probes also overlapped interchromosomal duplicons (Table The Illumina Beadchip (Human WG) also showed uneven coverage in the 15q11.2q13 region  and genomic positions (hg18) of the forward and reverse primer pairs.Click here for file


In 2011, the Tumour Node Metastasis (TNM) staging system still remains the gold standard for stratifying colorectal cancer (CRC) patients into prognostic subgroups, and is considered a solid basis for treatment management. Nevertheless, there is still a challenge with regard to therapeutic strategy; stage II patients are not typically selected for postoperative adjuvant chemotherapy, although some stage II patients have a comparable outcome to stage III patients who, themselves do receive such treatment. Consequently, there has been an inundation of ‘prognostic biomarker’ studies aiming to improve the prognostic stratification power of the TNM staging system. Most proposed biomarkers are not implemented because of lack of reproducibility, validation and standardisation. This problem can be partially resolved by following the REMARK guidelines. In search of novel prognostic factors for patients with CRC, one might glance at a table in the book entitled ‘Prognostic Factors in Cancer’ published by the International Union against Cancer (UICC) in 2006, in which TNM stage, L and V classifications are considered ‘essential’ prognostic factors, whereas tumour grade, perineural invasion, tumour budding and tumour-border configuration among others are proposed as ‘additional’ prognostic factors. Histopathology reports normally include the ‘essential’ features and are accompanied by tumour grade, histological subtype and information on perineural invasion, but interestingly, the tumour-border configuration  and especially tumour budding are rarely reported. Although scoring systems such as the ‘BRE’ in breast and ‘Gleason’ in prostate cancer are solidly based on histomorphological features and used in daily practice, no such additional scoring system to complement TNM staging is available for CRC. Regardless of differences in study design and methods for tumour-budding assessment, the prognostic power of tumour budding has been confirmed by dozens of study groups worldwide, suggesting that tumour budding may be a valuable candidate for inclusion into a future prognostic scoring system for CRC. This mini-review therefore attempts to present a short and concise overview on tumour budding, including morphological, molecular and prognostic aspects underlining its inter-disciplinary relevance. The REMARK guidelines proposed in 2005 try to improve the quality of biomarker studies by suggesting a strict study design  patients is ideally multidisciplinary and includes the fields of oncology, surgery, radio-/oncology, gastroenterology and pathology. The pathologist's main role is to provide an optimal histopathology report including all information that allows the clinician to design the best therapeutic strategy for the patient. According to the International Union against Cancer's (UICC's) publication ‘Prognostic Factors in Cancer’, Tumour Node Metastasis (TNM) stage, as well as L and V classifications are considered ‘essential’ prognostic factors, whereas tumour grade, tumour budding, tumour-border configuration, perineural invasion, medullary type, CEA and perforation are considered ‘additional’ prognostic factors . AlthougAn interesting observation is that in contrast to breast and prostate cancer where the ‘BRE’ and ‘Gleason’ score are used in daily routine, there has been no real advancement with respect to additional prognostic factors or scoring systems in CRC. Although tumour grade is consistently reported, it is a feature which suffers from poor interobserver reproducibility and does not have the expected impact on prognosis . In searTumour budding reflects a detachment of tumour cells at the invasive front of CRC into single cells or clusters up to five cells , which allows a polarised cell to assume a more mesenchymal phenotype with increased migratory capacity, invasiveness, resistance to apoptosis and production of extracellular matrix (ECM) molecules . Althougβ-catenin within the nucleus rather than in cytoplasm or membrane in tumour-budding cells, as well as increased laminin 5 gamma 2 and activation of SLUG and ZEB1, but also express fibronectin within the cytoplasm, implying a more mesenchymal phenotype underlining the interaction between tumour buds and the surrounding stroma . Activatβ(III)-tubulin and CXCL12  in high-grade tumour-budding cases . This prd CXCL12 .Over the last 5 years, the number of publications investigating tumour budding as a predictor of lymph node positivity, local and distant relapse, lymphovascular invasion and poor prognosis among patients of all pathological stages has markedly increased. In fact, tumour budding is now considered a category IIB prognostic factor . Tumour et al conducted a study on 663 patients showing first that tumour budding led to worse outcome and second, that Dukes' B patients with tumour budding had similar, if not worse, survival than Dukes' C patients with no budding (vs specific immune cell types, may be more important than the contribution of either separately (Tumour budding is a factor of poor prognosis, and in most studies, independent of pathological stage. In 1993, Hase Although tumour budding is typically thought of as a diagnosis restricted to the invasive front of CRC, The growing body of evidence supports tumour budding as an aggressive and adverse prognostic factor in all pathological stages of disease (In daily practice, a pathology report should include information on TNM, L and V, tumour grade and the histological subtype . Many stOn the basis of experience with other reported histomorphological features in various tumour types such as the mitoses count in breast cancer, soft tissue tumours and gastrointestinal stromal tumours, a reliable method for scoring such features is the 10 high-power fields average . Indeed,In summary, tumour budding should be considered a promising and strong prognostic factor in CRC. Its definite implementation will depend on a selected, internationally accepted scoring system. Additionally, based on functional studies, tumour buds could be a potential target for new therapeutic approaches.


In phase II cancer trials, tumour response is either the primary or an important secondary endpoint. Tumour response is a binary composite endpoint determined, according to the RECIST criteria, by 1) whether the percentage change in tumour size is greater than a prescribed threshold and 2) (binary) criteria such as whether a patient develops new lesions.Further binary criteria, such as death or serious toxicity, may be added to these criteria. The probability of tumour response (i.e. `success' on the composite endpoint) would usually be estimated simply as the proportion of successes among patients. This approach uses the tumour size variable only through a discretised form, viz. whether or not it is above the threshold. In this presentation, we discuss a method which also estimates the probability of success but which gains precision by using the information on the undiscretised (i.e. continuous) tumour size variable. This approach can also be used to increase the power to detect a difference between the probabilities of success under two different treatments in a comparative trial. We demonstrate these increases in precision and power using simulated data.We also apply the method to real data from a phase II cancer trial, and show that it results in a considerably narrower confidence interval for the probability of tumour response. Extending the work to other endpoints used in phase II trials, such as progression-free survival will be discussed.


Inflammasomes sense infection and cellular damage and are critical for triggering inflammation through IL-1β production. In carcinogenesis, inflammasomes may have contradictory roles through facilitating antitumour immunity and inducing oncogenic factors. Their function in cancer remains poorly characterized. Here we show that the NLRP3, AIM2 and RIG-I inflammasomes are overexpressed in Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC), and expression levels correlate with patient survival. In tumour cells, AIM2 and RIG-I are required for IL-1β induction by EBV genomic DNA and EBV-encoded small RNAs, respectively, while NLRP3 responds to extracellular ATP and reactive oxygen species. Irradiation and chemotherapy can further activate AIM2 and NLRP3, respectively. In mice, tumour-derived IL-1β inhibits tumour growth and enhances survival through host responses. Mechanistically, IL-1β-mediated anti-tumour effects depend on infiltrated immunostimulatory neutrophils. We show further that presence of tumour-associated neutrophils is significantly associated with better survival in NPC patients. Thus, tumour inflammasomes play a key role in tumour control by recruiting neutrophils, and their expression levels are favourable prognostic markers and promising therapeutic targets in patients. Inflammasomes are multiprotein complexes consisting of the pattern recognition receptors NLRP3, NLRC4, AIM2 or RIG-I; the adaptor protein ASC; and caspase-1. Inflammasome assembly leads to caspase-1 activation, which cleaves intracellular pro-IL-1β into secretable IL-1β. Inflammasomes are activated by pathogen-associated molecular patterns (PAMPs) including viral DNA . The less significant p-values of RIG-I, NLRP3, NLRC4 and NLRP7 are likely due to the smaller sample size analysed in this Q-PCR study. We nevertheless included RIG-I (2.212-fold), NLRP3 (3.645-fold), NLRC4 (2.040-fold) and NLRP7 (3.367-fold) for further study.We analysed the expression of inflammasome components in NPC tumours and adjacent normal tissues using Affymetrix microchips  or DAMPs present in the tumour microenvironment (ATP and ROS), or could be affected by the caspase-1 inhibitor, Z-VAD-FMK or depletion of inflammasome components by RNA interference. After screening four NPC cell lines for expression of key inflammasome components , we chosbiopsies , and infbiopsies  were usebiopsies . We founbiopsies . Howeverbiopsies .in vitro synthesized-EBER1 and EBER2, and secretion was inhibited by Z-VAD-FMK (2O2). IL-1β was significantly stimulated by ATP and H2O2, and this effect could be completely blocked by Z-VAD-FMK , were chosen to generate B16F10-IL-1β, B16F10-pro-IL-1β (expressing non-secretable IL-1β) and B16F10-vector cells  to obtain low to high levels of secreted cytokine  than those of B16F10-vector cells (376 mm2). Our data showed that the threshold for the antitumour activity of IL-1β was 64.2 pg/ml (Since IL-1β does not directly inhibit tumour cell proliferation (Supporting Information p = 0.003) or B16F10-vector  cells (p = 0.034) or B16F10-vector  cells  were significantly better than for mice carrying tumours arising from B16F10-pro-IL-1β  cells . We furt1) cells . Collect1) cells . Thus, oIl1r1−/− mice  indeed had a clear neutrophil-like morphology  and protumour N2 TANs (immunosuppressor)  in TANs from B16F10-IL-1β tumour-bearing mice was noted 2 weeks after treatment (p = 0.006) as compared to B16F10-IL-1β tumour-bearing mice treated with isotope antibody. In contrast, depletion of neutrophils in B16F10-vector tumour-bearing mice did not affect tumour growth. Together, these data indicate that depletion of TANs resulted in significant abrogation of the IL-1β-mediated antitumour activity, suggesting that TANs contribute to the antitumour activity of tumour-derived IL-1β.We next studied the functional significance of TANs in IL-1β-mediated tumour control by depleting the Ly6Greatment . PalpablWe further analyzed the significance of TANs on tumour development in NPC patients. Thirteen out of the 140 tested NPC specimens were TAN-positive as shown by hematoxylin and eosin (H&E) staining . Kaplan–p = 0.043; Supporting Information In order to delineate the function of inflammasomes in other cancer types, we evaluated the expression patterns of AIM2, RIG-I, NLRP3, ASC and caspase-1 in 114 cancer cell lines involving 22 cancer types. For this analysis, we used 324 Affymetrix U133 Plus 2.0 array data sets from the eight NPC cell lines generated in this study and 106 cell lines from the public GSK Cancer Cell Line Genomic Profiling Data  in the blood stream and poor survival was recently reported in NPC  and the NIH Belmont Report (http://ohsr.od.nih.gov/guidelines/belmont.html). Patients diagnosed with distant metastatic disease at presentation (M1 stage) and/or those who had undergone previous treatment at another institute were excluded from the present study. Patient characteristics and clinical features are summarized in Supporting Information The retrospective cohort comprised 144 NPC patients who had been admitted to Chang Gung Memorial Hospital (CGMH) at Lin-Kou from 1990 to 2000. Clinical stage was defined according to the 2002 cancer staging system revised by the American Joint Committee on Cancer. Histological typing was done according to the World Health Organization (WHO) classification criteria , oATP, apyrase, DPI, CA-074 Me, Z-VAD-FMK and cisplatin were purchased from Sigma–Aldrich and H2. HK1-EBV cells were maintained with 500 µg/ml G418. Lentiviruses were established by following the protocol of the RNAi Core, Taiwan (http://rnail.genmed.sinica.edu.tw), and used to generate human IL-1β-expressing or vector-control HK1 cells, as well as mouse pro-IL-1β-expressing, mouse IL-1β-expressing or vector-control B16F10 cells. For transfection assays, sonicated EBV gDNA (around 500 bp), poly (dA:dT), pEGFP, EBER1/2, pHEBo, pHEBo-EBER1/2 were transfected by Lipofectamine 2000 (Invitrogen). For irradiation treatment, HK1 cells were irradiated at 10, 20, 30 or 40 Gy using Gammacell 3000 Elan (MDS Nordion) at 5.0 Gy/min.NPC-TW01, -TW02, -TW04 and HK1 cells were cultured under conditions described before  with 1 × 105 tumour cells. Both survival and local tumour growth were determined twice weekly. For the surgery treatment, the tumours were established with 1 × 106 cells by i.f.p. injection and the tumour-bearing leg was amputated below the knee at day 14. For FACS, RNA and cell subset isolation studies, tumours were harvested from the mice, and digested with 60 µg/ml DNase I (Sigma–Aldrich) and 1 mg/ml collagenase D (Roche) at 37°C for 20 min. All experiments were repeated at least twice.Mouse experiments were performed under the approval of the Institutional Animal Care and User Committee of Chang-Gung University. C57BL/6 and nude mice  were obtained from National Laboratory Animal Center, Taiwan. +/Ly6G+ neutrophils were sorted using a FACSAria cell sorter (BD Biosciences). Fluorescently labelled antibodies were listed in Supporting Information Tumour cells were analysed by FACSCalibur flow cytometer and CellQuest pro software (BD Biosciences). The CD11b+/Ly6G+ cells from tumours were collected using a Shandon Cytospin 4  at 800 rpm for 5 min and stained with Liu's stain solution .The sorted CD11bPROBLEM:Inflammasomes sense infection and cellular damage, and are critical for triggering inflammation through IL-1β production. To date, there has been no study on the roles of inflammasomes and their clinical implications in virus-associated human cancers. Inflammasomes are activated by two major groups of activators, pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), which are potentially present in the tumour microenvironment of infection-associated cancers. However, the expression profile of inflammasomes in malignant cells remains unclear. NPC is closely associated with EBV infection. The current treatment remedies are irradiation and cisplatin; however, local recurrence after the treatment remains a major clinical problem. In addition, the link between inflammasome function in NPC cells, on the one hand, and EBV infection- and stress-induced inflammation and cancer, on the other, is unknown.RESULTS:in vitro and in vivo. AIM2, RIG-I and NLRP3 inflammasomes in NPC tumour cells, as well as tumour-associated neutrophils were identified as new markers for favourable prognosis in NPC. Inflammasomes in tumour cells can be activated by tumour microenvironmental factors including microbial products  and tumour-derived danger signals (ATP and H2O2). Importantly, the therapeutic treatments, irradiation and cisplatin, can activate AIM2 and NLRP3 inflammasomes, respectively, and further enhance IL-1β production in the presence of tumour microenvironmental factors. Tumour-derived IL-1β can mediate anti-tumour activity by recruiting immunostimulatory neutrophils, which function as the effector cells for the inhibitory effect of tumour-derived IL-1β on tumour growth. In addition, high expression of AIM2, RIG-I and NLRP3 inflammasomes is significantly correlated in EBV-positive lymphoma cell lines, suggesting that EBV infection is a potential factor for inflammasome overexpression in cancer cells.We addressed the problem by carrying out comprehensive analyses of inflammasomes using clinical samples and mechanistic studies IMPACT:This is the first study to establish the roles of inflammasomes in tumour cells responding to tumour microenvironmental factors and therapeutic treatments, and contributing to tumour-associated neutrophil-dependent tumour suppression. The elevated expression of NLRP3, AIM2 and RIG-I inflammasomes and IL-1β in malignant cells and tumour-associated neutrophils can be useful as prognostic biomarkers in NPC patients and may be important targets for developing treatments in EBV-associated cancers.Neutrophils were depleted by using 100 µg of purified monoclonal anti-Ly6G antibody 1A8 or isotype-matched control IgG (BD Biosciences) via intraperitoneal injections (i.p.) starting 18 days after inoculation with tumour cells, and followed by i.p. injection twice a week throughout the entire experimental period. Tumour neutrophil depletion was confirmed by flow cytometry.Immunoprecipitation and Western blotting have been previously described . The primers used for cloning are listed in Supporting Information EBER1 and EBER2 cDNAs generated from C666-1 NPC cells by reverse transcription were cloned into pGEMTeasy (Promega). Purified PCR products of T7 promoter-fused EBER1 or EBER2 were used as a template of RNA interference has been previously described  and mouse IL-1β (R&D Systems).4 cells were seeded, and total cell numbers were counted each day for 4 days.5 × 10The LMP1 constructs and transfection methods have been described previously (Chen et al, In vitro data and tumour growth in mouse were analysed with the two-tailed Student's t test. Differences were considered significant at p < 0.05. The error bars were calculated and represented in terms of mean ± SD.Statistical analyses were performed using the SPSS 13.0 statistical software package. Clinicopathologic features were evaluated using the Pearson Chi-Square test. Survival curves were plotted using the Kaplan–Meier method and compared using the log-rank test. The Cox proportional hazards model was applied for multivariate analysis. The cut-off values to define high intensity of IHC staining in tumour cells were determined from Receiver Operating Characteristics curve analysis.


Measurement of genome-wide DNA methylation (DNAm) has become an important avenue for investigating potential physiologically-relevant epigenetic changes. Illumina Infinium  is a commercially available microarray suite used to measure DNAm at many sites throughout the genome. However, it has been suggested that a subset of array probes may give misleading results due to issues related to probe design. To facilitate biologically significant data interpretation, we set out to enhance probe annotation of the newest Infinium array, the HumanMethylation450 BeadChip (450 k), with >485,000 probes covering 99% of Reference Sequence (RefSeq) genes , Bethesda, MD, USA). Annotation that was added or expanded on includes: 1) documented SNPs in the probe target, 2) probe binding specificity, 3) CpG classification of target sites and 4) gene feature classification of target sites.in silico. Measurements from these non-specific probes likely represent a combination of DNAm from multiple genomic sites. The expanded biological annotation demonstrated that based on DNAm, grouping probes by an alternative high-density and intermediate-density CpG island classification provided a distinctive pattern of DNAm. Finally, variable enrichment for differentially methylated probes was noted across CpG classes and gene feature groups, dependant on the tissues that were compared.Probes with documented SNPs at the target CpG (4.3% of probes) were associated with increased within-tissue variation in DNAm. An example of a probe with a SNP at the target CpG demonstrated how sample genotype can confound the measurement of DNAm. Additionally, 8.6% of probes mapped to multiple locations DNAm arrays offer a high-throughput approach for which careful consideration of probe content should be utilized to better understand the biological processes affected. Probes containing SNPs and non-specific probes may affect the assessment of DNAm using the 450 k array. Additionally, probe classification by CpG enrichment classes and to a lesser extent gene feature groups resulted in distinct patterns of DNAm. Thus, we recommend that compromised probes be removed from analyses and that the genomic context of DNAm is considered in studies deciphering the biological meaning of Illumina 450 k array data. Measuring epigenetic marks has become an attractive approach for connecting phenotype, genetics and environment in many fields of medicine -3. DNA mhttp://genome.ucsc.edu; UCSC Genome Bioinformatics, Santa Cruz, CA, USA), CpG islands and shores, and regulatory features. However, recently technical limitations of the Infinium platform have been described . ,33. 7,33Previous studies have shown that tissue-specific differences in DNAm occur in CpG island shores . We wereA goal of the additional CpG classification of 450 k probes was to identify biologically-relevant structures that might underlie genome-wide changes in DNAm. The HIL CpG classes demonstrated a more extreme DNAm profile and larger percentage of tDM probes which may be reflective of biological processes. Intriguingly, even though ICs and ICshores have the same CpG density, distinct differences between these two classes emerged in our analyses, suggesting that ICs that border HCs are distinct from ICs on their own, highlighting the utility of this additional classification.There is increasing evidence that DNAm of gene features outside of CpG islands and promoters may be an important marker of gene expression. For example, it has been shown that DNAm of the first exon is correlated with transcriptional repression . CoveragDue to the observed differences in DNAm across HIL CpG classes detailed in the previous section, gene features were further subclassified by HIL CpG class. Given the known bias in the distribution of CpGs in the genome , there wWe were also interested in assessing where tissue-specific differences in DNAm occurred based on gene features. Thus, we examined tDM probes for enrichment within each gene feature group  under the accession [GSE:42409]. Based on our findings, we suggest that all 450 k users analyze data with the following factors in mind. Probe signals may be biased by the presence of SNPs in the target CpG and/or binding of probes to multiple genomic locations. SNPs at the target CpG may be especially problematic in studies with small sample size, as chance may result in dramatic differences in the frequency of polymorphisms between groups. However, false positives may still result in studies with larger sample sizes, if groups are not ethnically matched. Additionally, DNAm patterns within CpG enrichment classes or gene features could overshadow findings between study groups if probes are not separated and considered within these genomic features.In this article, we presented an expanded annotation of the 450 k array including both compromised probe annotation and additional biologically-relevant annotation. Our expanded annotation has been deposited as a platform on the NCBI GEO  because of the potential to bias measurement of DNAm . Furtherw/o CpG) and 3) the interval of the entire probe (entire probe) : 1) the interval of the target CpG (CpG), 2) the interval containing the probe but excluding the target CpG  from UCSC . Probes with a detection p value >0.01 in any sample, probes with no ß value in any sample, all rs and ch probes, all sex chromosome and non-specific probes were removed prior to analyses. The level of DNAm for 428,216 probes in our sample dataset was intersected with the expanded annotation for further analyses.Chips were scanned using an Illumina HiScan on a two-color channel to detect Cy3 labeled probes on the green channel and Cy5 labeled probe on the red channel. Illumina GenomeStudio Software 2011.1 was used to read the array output and conduct background normalization. The signalA, signalB and probe intensity were exported for autosomal probes and read into R. M values were generated using the Bioconductor . Primer sequences and probe information are available in Additional file p value cutoff of 0.05 was selected with a Bonferroni correction to account for repeated comparisons [p values <1.0 × 10-7. All figures were created in R and Adobe Illustrator CS6.A KS test was used to assess the difference in distribution of SD in ß values for probes that contained SNPs. The KS statistic represents the maximum absolute difference between the cumulative distributions of two functions. Probes with small within-tissue SD in ß (<0.10) were removed from all probe groups to increase the power of the analysis. Probes with a target CpG SNP were removed from the SNP <10 bp group. The number of probes included in the SD in ß distribution curves for blood samples was 5,450 for all probes, 809 for SNP >10 bp, 402 for SNP <10 bp and 2,190 for target CpG SNP, and for the aging dataset was 6,267 for all probes, 1,022 for SNP >10 bp, 362 for SNP <10 bp and 2,753 for target CpG SNP. KS tests were also used to assess the difference in distribution of DNAm between Illumina CpG classes and between HIL CpG classes. Fisher’s exact test was used to compare the distribution of the number of probes within the three levels of DNAm for both Illumina and HIL-annotated CpG classes: hypomethylated , heterogeneously methylated  and hypermethylated . Enrichment analyses of tDM probes were performed in Python (Python Software Foundation). To select tDM probes, DNAm was first averaged for each probe within a tissue. A z-score was calculated for each probe comparison between tissues. A 27 k: Infinium HumanMethylation27k BeadChip; 3’UTR: 3’ untranslated region; 450 k: Infinium HumanMethylation450 BeadChip; 5’UTR: 5’ untranslated region; auto: autosomal; bp: base pair; chrs: chromosomes; CNV: copy-number variation; dbSNP: database of single nucleotide polymorphisms; DNAm: DNA methylation; EWAS: epigenome-wide association studies; FDR: false discovery rate; GEO: Gene Expression Omnibus; GWAS: genome-wide association studies; HC: high-density CpG island; HIL: high-density CpG island (HC), intermediate-density CpG island (IC) and non-island (LC) or low-density regions; IC: intermediate-density CpG island; ICshore: intermediate-density CpG island shore; KS: Kolmogorov-Smirnov; LC: non-island; mSNP: methylation-associated SNP; NCBI: National Center for Biotechnology Information; Obs/Exp: Observed/Expected; PBMC: peripheral blood mononuclear cell; PCR: polymerase chain reaction; RefSeq: Reference Sequence; SAM: significance analysis of microarrays; SNP: single nucleotide polymorphism; SWAN: subset-quantile within array normalization; tDM: differentially methylated between tissues; TSS: transcription start site; UCSC: University of California, Santa Cruz.The authors declare that they have no competing interests.EMP carried out Pyrosequencing, conducted SNP annotation and analysis, participated in CpG island annotation and analysis, participated in the design of the study, and drafted the manuscript. AMC participated in CpG island annotation and analysis, participated in the design and analysis of the study, and conceived of the study. LLL processed Illumina data, conducted gene feature annotation and participated in the design of the study. PF conducted enrichment analyses. EE conducted non-specific probe analyses and participated in the design of the study. MSK and CJB conceived of the study, participated in data analysis and participated in the design of the study. WPR participated in data analysis and design of the study. All authors contributed to the writing of the manuscript, and read and approved the final version.Relative location of probes to target CpG. To complete our analysis, it was necessary to locate 450 k probes within the genome. Illumina annotated the hg19 location of each target C  and the strand on which the probe was designed; R probes bind to the negative strand, whereas F probes bind to the positive strand. With this information we annotated the start and end coordinates for all probes on the array. Refer to Additional file 14 for the start and end location for each probe type. Type I versus type II probes and F versus R probes align differently with target CpGs. Single nucleotide extension of a probe occurs by one of four fluorescently labeled nucleotides, A and T are labeled in red, while C and G are labeled in green. The color of single nucleotide extension of type I probes is not dependant on whether the target site is methylated or unmethylated; however, for type II probes, incorporation of an A or T signals an unmethylated site in red and the incorporation of a C or a G signals a methylated site in green.Click here for fileFrequency of target SNP CpG/probe.Click here for fileDistribution of DNAm at three highly variable probes. The level of DNAm was plotted for three highly variable probes (SD in ß ≥0.25) annotated with a target CpG SNP, across the 261 individuals in the aging dataset. cg06961873 corresponds to the CpG site genotyped in Figure 1D. A trimodal pattern of DNAm was observed at these three exemplary sites, indicating that DNAm measured at these sites may reflect sample genotype.Click here for fileList of autosomal probes with sex differences in DNAm.Click here for fileIllustration of Illumina and HIL CpG classes. (A) Diagram of Illumina-annotated probes, based on their relative location to a CpG island: within the island, shore or shelf. We used the term ‘sea probes’ to refer to probes that were not annotated into one of the Illumina CpG classes. Islands were defined based on UCSC criteria: CG content >50%, Obs/Exp, CpG ratio >0.60 and length >200 bps. Shores were defined as the 2 kb up- and down-stream of a CpG island and shelves as the 2 kb outside of a shore. (B) The HIL definition of CpG islands was used to annotate probes into three CpG classes: HC probes (map to a high-density CpG island or HC), IC probes (map to an isolated intermediate-density CpG island or IC) and ICshore probes (map to a region with IC density that borders an HC). The remainder of probes did not map to a CpG island and were thus considered non-island or LC probes. HCs were defined as CG content >55%, Obs/Exp CpG ratio >0.75 and length >500 bps, while ICs were defined as CG content >50%, Obs/Exp CpG ratio >0.48 and length >200 bps.Click here for fileDistribution of probes within Illumina-annotated and HIL-annotated CpG classes.Click here for fileDistinct patterns of DNAm across Illumina-annotated and HIL-annotated CpG classes in blood. Density curves were plotted using average ß values for probes within each Illumina-annotated and HIL-annotated CpG class in blood (n = 4). The number of probes contributing to each curve was: island = 136,712, shore = 100,083, shelf = 39,833, sea = 151,588, HC = 139,826, ICshore = 100,164, IC = 30,467 and LC = 157,759. For Illumina-annotated CpG classes, KS statistics in comparison to the distribution of DNAm of sea probes was 0.67 for island probes, 0.34 for shore probes and 0.06 for shelf probes. For HIL-annotated CpG classes, KS statistics in comparison to the distribution of DNAm of LC probes was 0.77 for HC probes, 0.53 for ICshore probes and 0.08 for IC probes.Click here for fileDistinct patterns of DNAm across Illumina-annotated and HIL-annotated CpG classes in buccal samples. Density curves were plotted using average ß values for probes within each Illumina-annotated and HIL-annotated CpG class in buccal samples (n = 4). The number of probes contributing to each curve was: island = 136,712, shore = 100,083, shelf = 39,833, sea = 151,588, HC = 139,826, ICshore = 100,164, IC = 30,467 and LC = 157,759. For Illumina-annotated CpG classes, KS statistics in comparison to the distribution of DNAm of sea probes was 0.66 for island probes, 0.32 for shore probes and 0.06 for shelf probes. For HIL-annotated CpG classes, KS statistics in comparison to the distribution of DNAm of LC probes was 0.76 for HC probes, 0.49 for ICshore probes and 0.07 for IC probes.Click here for fileDistinct patterns of DNAm across Illumina-annotated and HIL-annotated CpG classes in chorionic villi. Density curves were plotted using average ß values for probes within each Illumina-annotated and HIL-annotated CpG class in chorionic villi (n = 4). The number of probes contributing to each curve was: island = 136,712, shore = 100,083, shelf = 39,833, sea = 151,588, HC = 139,826, ICshore = 100,164, IC = 30,467 and LC = 157,759. For Illumina-annotated CpG classes, KS statistics in comparison to the distribution of DNAm of sea probes was 0.61 for island probes, 0.28 for shore probes and 0.08 for shelf probes. For HIL-annotated CpG classes, KS statistics in comparison to the distribution of DNAm of LC probes was 0.72 for HC probes, 0.45 for ICshore probes and 0.10 for IC probes.Click here for fileDistribution of ß values within Illumina-annotated and HIL-annotated CpG classes for blood.Click here for fileEnrichment of differentially methylated probes in Illumina-annotated and HIL-annotated CpG classes.Click here for fileAverage DNAm and SD of nine gene features.Click here for fileEnrichment of tDM probes within gene features.Click here for fileCalculation of intended genomic location of 450k probes.Click here for fileUCSC track of 450 k target Cs.Click here for fileUCSC track of HC/IC CpG islands.Click here for filePrimers for genotyping validation of a target CpG SNP.Click here for file


Renal replacement therapy is frequently required in critically ill patients with acute kidney injury. With intermittent hemodialysis, large volumes of fluid need to be removed over a relatively short period of time, jeopardizing hemodynamic stability in already hemodynamically compromised patients. Established methods of dry weight estimation are not practical in critical care and the estimation of excess body fluid removable by hemodialysis constitutes a particular change in these patients. Dynamic parameters of fluid responsiveness are increasingly being used to guide fluid therapy in critical care, but their suitability to monitor fluid removal with hemodialysis is not known.The aim of our study was to analyze changes in a dynamic parameter of fluid responsiveness (pulse pressure variation) in critically ill patients submitted to intermittent hemodialysis.Changes in pulse pressure variation, central venous pressure, median arterial pressure, and cardiac index were analyzed every hour over intermittent hemodynamics using a minimally hemodynamic monitoring device (LIDCO plus) in 28 mechanically ventilated patients. Additional measurements of lactate and central venous saturation were measured at the same time.P = 0.78) and central venous saturation  were not significantly different, but at the end of the procedure a significant difference in lactate was observed .Median dialysis duration was 4.5 hours, and a median of 2,900 ml fluid was removed. There were 102 hypotensive episodes. The median arterial blood pressure was 72 mmHg. Median CVP was 16 ± 6 and pulse pressure variation was 9 ± 6 just before hemodialysis. There was a significant increase in the pulse pressure variation over the dialysis treatment (15 ± 4) and a decrease in the CVP value (13 ± 6). Comparing the group of patients already fluid responsive (ΔPp >13%) just before the start of hemodialysis with the group nonfluid reponsive (ΔPp <13%), the median values of lactate  just before hemodialysis, the impact of fluid removal at the end of the procedure in perfusion parameters was significantly higher. Dynamic parameters of volemia could be useful to guide fluid removal and avoid hypoperfusion in acute renal failure patients mechanically ventilated during hemodialysis treatment.


Despite recent advances in treatment, lung cancer accounts for one third of all cancer-related deaths, underlining the need of development of new therapies. Mesenchymal stem cells (MSCs) possess the ability to specifically home into tumours and their metastases. This property of MSCs could be exploited for the delivery of various anti-tumour agents directly into tumours. However, MSCs are not simple delivery vehicles but cells with active physiological process. This review outlines various agents which can be delivered by MSCs with substantial emphasis on TRAIL (tumour necrosis factor-related apoptosis-inducing ligand). Lung cancer is one of the leading causes of mortality and morbidity in the world and accounts for one third of all the cancer-related deaths . An estiMesenchymal stem cells (MSCs) are a type of bone marrow-derived stem cell, which can differentiate in vitro into osteoblasts, chondrocytes and adipocytes. They do not possess any unique markers for their identification, so their identification relies on the expression of CD73, CD90 and CD105 while lacking CD34, CD45 and other haematopoietic stem cell markers [MSCs lack the expression of MHC II and its co-stimulatory molecules CD80 and CD86 and CD40 . This paIt has been widely demonstrated that MSCs home to and infiltrate into areas of new stroma formation possibly forming crucial stromal support . This haThe precise mechanism of homing of MSCs to the tumours is not fully mapped, but it was widely accepted that the chemokines released by the tumours attract MSCs. This is substantiated by the presence of a wide variety of chemokine receptors on the MSC cell surface and experiments in vitro and in mouse models that have either over- or under-expressed these receptors, showing a change in MSC homing capabilities ,16,17,18CXCL12 and its receptor CXCR4 have generated particular interest in MSC homing. Their knockouts are universally fatal in utero and their role in migration of haematopoietic cell migration is well characterised ,20. SeveWork on MSC homing is complicated and varying results may be explained by a number of factors. MSCs are extracted from various tissues and their lack of unique identification markers to classify them results in a number of different populations being used making cross-referencing results difficult. Furthermore, different in vitro culture conditions and the passage numbers used alter the expression of cell surface receptors ,25. ThisThe homing capability of MSC can be exploited to deliver pro-apoptotic agents straight into the tumour micro-environment. Several studies have achieved this with varying success and are summarised in table The majority of studies have used MSCs engineered to express and deliver a variety of cytokines. Interleukin (IL)-2, an immune modulatory cytokine, has been shown when over-expressed by MSCs to improve immune surveillance against tumours and reduce metastasis from a subcutaneous model . SimilarAn exciting set of viruses which selectively target and inhibit tumour cells without affecting normal cells are termed oncolytic viruses . These vMSCs have also been engineered to express an enzyme which converts a pro-drug into a cytotoxic agent at the site of tumours. This has been successfully demonstrated in a glioma model  where MSThere is increasing interest in the use of nanoparticles in a variety of biomedical applications. However, the ability to deliver them efficiently to a disease that is systemically distributed remains a key challenge. Again, MSC tumour tropism has been used as a method of tumour targeting. A silica nanorattle-doxorubicin drug delivery system was efficiently anchored to MSCs by specific antibody targeting the CD90 receptor on the MSC cell surface and successfully delivered into a glioma model . InteresTRAIL (tumour necrosis factor-related apoptosis-inducing ligand) is the most studied and well-characterised pro-apoptotic agent widely accepted to be ideal as MSC cargo. TRAIL, also known as APO2 ligand, is a type II transmembrane protein with 281 amino acids and a member of the TNF death ligand superfamily. TRAIL triggers the extrinsic death pathway  and TRAIL-R2 (DR-5) are able to transduce a signal into the cell after binding of TRAIL to their extracellular domains. The receptors TRAIL-R3 and TRAIL-R4 do possess extracellular domains capable of binding to the ligand but lack an intracellular cytoplasmic domain, thus failing to mediate death signals to the intracellular apoptotic machinery. Hence, TRAIL-R3 and TRAIL-R4 act as decoy receptors that antagonize TRAIL-induced apoptosis. All these receptors form heterotrimers upon binding of the ligand. Osteoprotegerin is a soluble protein which possesses the capability of binding to TRAIL with low affinity . This prThe novelty of TRAIL is that it only induces apoptosis in transformed cells with virtually no effect on normal cells. This makes TRAIL a unique therapeutic with very few off-target adverse effects characteristic of chemotherapeutic agents and radiation. The mechanism for this selective targeting of tumour cells is not well characterised. The decoy receptor theory states that the normal cells express decoy receptors while the transformation of cells makes them express DR4 and DR5 thus making them vulnerable to TRAIL . HoweverThe selective tumour-specific cytotoxicity of TRAIL has led to hailing it as a ‘silver bullet’ for the treatment of cancer. However, its limited bioavailability and poor pharmacokinetic profile have made its use a serious challenge. The half-life of TRAIL is very short at around 30 min . To circHowever, not all tumours are fully sensitive to TRAIL. TRAIL triggers the extrinsic apoptotic pathway, while conventional chemotherapeutics and radiation trigger the intrinsic apoptotic pathway fig. . It woulIdentifying and targeting proteins responsible for TRAIL resistance may also increase the anti-tumour potency of TRAIL, such as cFLIP (cellular FLICE-inhibitory protein) ,67, cIAPThe ability of MSCs to home effectively to tumours makes them an attractive therapeutic option. However, MSCs are not merely vehicles which transport the therapies but are cells possessing physiological properties.It is widely accepted that in large numbers MSCs possess immunosuppressive effects in vitro. They are capable of arresting the immune cells in G0/G1 phases thus preventing the S-phase entry and subsequent cell division. This has been demonstrated in T cells , B cellsThese anti-inflammatory effects have been tested in a number of clinical trials in inflammatory conditions, such as inflammatory bowel disease and chronic obstructive pulmonary disease, and in other diseases, such as cardiac disease. Several studies show an enhanced cardiac function ,77,78,79The immunosuppressive capability of MSCs could, however, be a double-edged sword. Their immunosuppressive nature could potentially interfere with any physiological anti-cancer immune cell function in the tumour environment.Reports on untransduced MSC effects on tumour growth are mixed. The majority of work suggests MSCs not only home to tumours but also have intrinsic anti-tumour properties. MSCs alone lead to benefit in a murine glioma model , in our In specific context, however, MSCs can appear to be tumour promoting, which was demonstrated by tumour development after subcutaneous co-administration of MSCs with allogeneic melanoma cells producing tumours, while allogeneic melanoma cells seeded on their own are not capable of tumour induction . This efAs discussed earlier, MSCs do produce a wide array of chemokines, cytokines and growth factors fig. . They maAnother important and serious concern is the ambiguity that MSCs might themselves undergo malignant transformation. Karyotype abnormalities have been noticed after in vitro passage of murine MSCs ,91,92 anDeveloping a cellular therapy using MSCs as delivery vectors is the ultimate goal of this area of research and MSCs have exhibited the potential for clinical translation. MSCs can be easily isolated, cultured in flasks and genetically modified. Their allogeneic application confers them an added advantage of being a possible off-the-shelf therapeutic. Their ability to target metastasis and provide a local high concentration of their cargo makes them unique.Many phase I and II clinical trials involving MSCs for a variety of treatments were recorded in the largest clinical trial database, clinicaltrials.gov. The therapeutic areas include graft-versus-host disease, ischaemic cardiac disease, Crohn's disease and chronic obstructive pulmonary disease . HoweverMSC have the potential to be ideal delivery vectors for a variety of pro-apoptotic agents in treating cancers. The lack of knowledge of MSC physiology within the tumour environment is producing caution, and more robust studies characterising their homing mechanisms may improve proposed therapies. Indeed, simple questions, such as how many cells need to be given and when, remain unanswered. However, their role as an adjunct in patients with metastatic tumours looks hopeful.All authors declare no conflicts of interest.


The mini-fluid challenge is a widely used strategy to manage fluid loading in the ICU and OR. Although it might be a rational strategy, data on the mini-fluid challenge and its reliability are very limited. We investigated the value of changes in pulse contour cardiac output as a result of a mini-fluid challenge of 50 and 100 ml to predict fluid loading responsiveness.We measured the effects after the administration of 50, 100 and 500 ml bolus colloid infusions on CO (Modelflow (COm) and LiDCO (COli)), CVP and MAP in 21 patients on mechanical ventilation after elective cardiothoracic surgery. From the data we analysed the smallest volume that was predictive for the effects of 500 ml on cardiac output.COli and COm increased after 50, 100 and 500 ml fluid loading. Best results are observed for changes in COm after 100 ml fluid loading . A change in Modelflow CO of at least 4.3% has a sensitivity of 67% and a specificity of 100% after 100 ml fluid loading. Sensitivity is 60% and specificity 83% for a similar cutoff in CO measured with the LiDCO device after 100 ml fluid loading. In our patient population, MAP and COli did not predict responsiveness with more accuracy than mathematical chance. See Figure 1.Changes in pulse contour CO can be used in a mini-fluid challenge to assess fluid responsiveness in our postcardiac surgery patients.


One of the components of the Kangaroo Method (KM) is the adoption of the Kangaroo Position. The skin-to-skin contact and the vertical position the child adopts when in this position may provide sensorial, vestibular and postural stimuli for the newborn. The Kangaroo Position may encourage vestibular stimuli and a flexed posture of the limbs, suggesting the hypothesis that the Kangaroo Position may have an impact on flexor muscle tone. The effect of these stimuli on the motor features of the newborn has not been the subject of much investigation. No study has yet been conducted to determine whether the Kangaroo Position may progressively increase electromyographic activity or whether this increase persists until term-equivalent age. The aim of this study was to evaluate the effect of the Kangaroo Position on the electromyographic activity of preterm children.A follow-up study was carried out between July and November 2011 at the Instituto de Medicina Integral Prof. Fernando Figueira (IMIP), Recife-Brazil, using a sample of 30 preterm children. Surface Eletromyography (SEMG) was used to investigate the muscle activity of biceps brachii. The electromyographic readings were taken immediately before (0 h) and after 24 h, 48 h, 72 h, 96 h of application of the Kangaroo Position as well as at the term equivalent age in each baby. Electromyographic activity was analyzed using the Root Mean Square (RMS) and the mean values of the times were analyzed by way of analysis of variance for repeated measures and the Tukey test.Electromyographic activity of the biceps brachii varied and increased over the whole 96h period (RMS:0 h = 36.5 and 96 h = 52.9) (F(5.174) = 27.56; p < 0.001) and remained constant thereafter . The correlations between the corrected age and the values for electromyographic activity did not show any statistical significance.The Kangaroo Position leads to a growing increase in the electromyographic activity of preterm children’s biceps brachii after up to 96 h of stimulation and this response persists until at least the 21st day after this period. One of the most important current neonatal health care initiatives is the Kangaroo Method (KM), which is a care method intended for preterm low-weight newborns . There iThe effect of these stimuli on motor features of the newborn has not been the subject of much investigation -14. In vA previous study conducted in the city of Recife, Brazil using Surface Eletromyography (SEMG) found an increase in the electromyographic activity of flexor muscles in babies adopting the Kangaroo Position for 24 h and that this effect persists for 24 h thereafter . HoweverThe aim of this study was thus to determine the electromyographic profile of preterm newborns at different stages during adoption of the Kangaroo Position and term equivalent age.A follow-up type study was carried out between July and November 2011 at the Instituto de Medicina Integral Prof. Fernando Figueira (IMIP), in Recife, Brazil with 30 preterm newborns hospitalized at the Institute’s Kangaroo Unit.2 and has a ward with 22 beds for clinically stable preterm newborns  The newborns had to tolerate food, to breathe without the use of an apparatus and to weigh more than 1,250 grams.The IMIP’s Kangaroo Unit covers a surface-area of 600 mThe Kangaroo Unit provides medical and nursing services and also speech therapy and physiotherapy. In this unit, the newborns, when referred by the medical services, are evaluated and undergo an early stimulation program.Preterm newborns considered eligible for inclusion in the sample were those with a gestational age of 27 to 34 weeks and a corrected age of less than 35 weeks during the first electromyographic evaluation, who had not previously experienced the Kangaroo Position. The exclusion factors were: an Apgar of less than 7 in the 5th minute, a prior history of grade III or grade IV intracranial hemorrhage , seizures, congenital infection , malformations of the Central Nervous System  Central Nervous System infections , congenital cardiopathy, trauma during delivery  and gastro-esophageal reflux disease.All these inclusion and exclusion factors were assessed using data collected from the records of patients diagnosed by neonatologists from the IMIP’s Neonatal Intensive Care Unit and the Kangaroo Unit. Figure A convenient non-probabilistic sequential sample was obtained from the newborns. The sample comprised 30 newborns aged between 27 and 34 weeks (with a mean age of 31.21 ± 2.1) and a mean weight, in grams, of 1407.3 ± 432.1. Other information on the sample can be found in Table The sample size was calculated using a pilot study, which confirmed the standard deviation in differences in electromyographic responses before and after adoption of the 9.3 microvolt Kangaroo Position and an estimate of the difference between the means of the two situations equal to five microvolts. With an alpha error of 0.05 and power of 0.8, the resulting sample size was 29.2 (rounded up to 30) individuals.The project was submitted to the IMIP’s Ethics Committee for Research involving Human Beings and was approved (protocol no. 1902). The parents or guardians who agreed to participate signed a Term of Free and Informed Consent.400® electromyograph (Miotec Equipamentos Biomédicos – Brazil). A system of channels and self-adhesive 4.2-mm-diameter Ag/AgCl electrodes (Meditrace® 100) were used to connect the body to the system. The electromyograph was connected to a laptop with Miographic 2.0 software (Miotec Equipamentos Biomédicos – Brazil) to process the signal. The sampling frequency was 2000 Hz. The electromyograms were magnified 2000-fold.The electromyographic signal was obtained using a Miotool Readings were registered using two electrodes placed in the central portion of the muscle of the biceps brachii, between the motor point and the myotendinous junction, parallel to the muscle fibers, as recommended by the Surface Electromyography for the Non-Invasive Assessment of Muscles (SENIAM) project  and alsoThe electromyographic signal of the newborns was captured with the aid of a small cushion in the shape of a 30° angle wedge, called a wedge cushion. The electromyographic records were read when the newborns were in Brazelton state 4 or 5, inactive or active alert, respectively .The first electromyographic reading was taken before adoption of the Kangaroo Position (0 h). Immediately after taking the first reading, the children were placed for the first time in the Kangaroo Position. The Kangaroo Position adopted was that recommended by the Neonatal Unit, in which the newborn is positioned against the adult’s chest, face down, wrapped in a strip of flexible cloth. Subsequent readings were taken after 24 h, 48 h, 72 h, 96 h of the Kangaroo Position and, finally, at term-equivalent age (40 ± 1 weeks). On each of these occasions, two readings were taken, the first with the arm at rest and the second with the arm flexed in response to the Dubowitz maneuver . Each reFor the first 96 h the babies were kept in the Kangaroo Position for 8–12 hours per day. The babies were removed from the Kangaroo Position (and placed on a soft cushion) for short intervals when the mothers went to the restroom or the shower and during breastfeeding or other forms of feeding.During data collection, the researchers asked the Kangaroo Unit not to give the newborns physiotherapy. The newborns did not, therefore, undergo any kind of early motor stimulation during data collection, except for oral stimulation by speech therapists, when necessary.For signal analysis this was transformed into Root Mean Square (RMS) and normalized ,21. The per se as a confounding variable, correlation was made between corrected age and the electromyographic data before adoption of the Kangaroo position (0 hrs). The alpha error for rejection of the null hypothesis was 0.05.One-way analysis of variance for repeated measures was carried out to compare the means for electromyographic activity at different intervals. Tukey test for multiple comparisons was also performed to compare each two intervals of electromyographic records. To exclude the effect of the development of the child The mothers of the newborns had a mean age of 27.2 ±6.7 years. The clinical characteristics of the mothers and the clinical and biological characteristics of the newborns are presented in Table =27.56; p < 0.001). Multiple post hoc comparisons (Tukey test) showed that, compared with the reading for 0h, the RMS was higher at 48 h (p = 0.03), 72 h (p < 0.001) and 96 h (p < 0.001). Moreover, there was a growing increase in RMS up to 96 h, between the following intervals: from 0 h-48 h ; from 24 h-72 h ; from 48h-96 h ; and from 72 h-96 h .The RMS varied significantly throughout the study period: , 24 h (p < 0.001), 48 h (p < 0.001), and 72 h (p < 0.001). However, there was no significant difference compared to the reading for 96 h (p > 0.99).The correlation Figure  between The results of the present study show a significant increase in electromyographic activity of the bíceps brachii muscle in newborns who have been placed in the Kangroo Position. Furthermore this increase was found to be steady and constant. This finding is worthy of note, as it suggests that this position produces a functional alteration in the muscle involved in the anti-gravity posture system.A previous study showed that early psychomotor stimulation of preterm newborns from the first to fifth corrected month of life improves motor development and that the more stimulation there is, the larger the number of motor skills acquired in the supine, prostrate and sitting positions . LikewisAlthough these other studies have examined the effect of psychomotor or sensorial stimulation on the motor development of preterm newborns, they did not specifically investigate the influence of the Kangaroo Position on this development -26.Few studies have been conducted that examine the effect of the Kangaroo Position on motor development -14. BarrIt may be that the stimulation of the kinesthetic and vestibular systems provided by the Kangaroo Position influenced the motor response observed in the aforementioned studies. The hypothesis that the vestibular and kinesthetic systems are involved in this process is backed up by the results presented in meta-analyses of interventions other than the Kangaroo Position ,26.Studies that showed the influence of the Kangaroo Position on the motor skills of newborns also found that this effect is persistent ,14. FerbAlthough we cannot rule out the possibility that the growing increase in electromyographic activity observed in the first 96 h of the Kangaroo Position may be due to the natural growth of the child, this is implausible, since there were no correlations between the ages of the newborns or their birth-weight and the level of electromyographic activity. Moreover, the results show that, even after an interval of 21 days between being discharged from hospital and the electromyographic reading at term equivalent age, the electromyographic responses at this point were similar to those found by the reading at 96 h.Furthermore, since the newborns did not undergo any motor intervention during the data collection period, the results show that the Kangaroo position is associated with the electromyographic responses found.One limitation of the present study may be the fact there was no comparison with a control group of newborns not placed in the Kangaroo Position, which might have revealed that the increase in RMS is not related to the Kangaroo Position but the result of the natural development/growth of the child. This limitation, however, is minimized by the lack of correlation between the age and the electromyographic activity.Another limitation is the fact that the results cannot be generalized for newborns with a gestational age of under 27 weeks and it is not known whether the RMS would increase, were the period of adoption of the Kangaroo position shorter than that used for the present study.The present study is, so far as we are aware, the first follow-up study of the effect of the Kangaroo Position on electromyographic activity in preterm children. Given the results found, it would be particularly interesting to investigate other muscles involved in the postural system, and to conduct a study similar to this one with term children and children who have not been placed in the Kangaroo Position.In short, the results of the present study provide further evidence that the Kangaroo Position causes an increase in the electromyographic activity of the biceps brachii in preterm newborns, increasing over 96 h and persisting until term equivalent age. Given that the electromyographic test was carried out on the muscle at rest, the increase in this myoelectric activity may suggest an increase in flexor muscle tone in the children, at least in the case of the muscle studied. It could therefore be that the Kangaroo Position provides not only physiological and behavioral benefits, as shown in other studies, but may also be associated with alterations in muscle function, improving the posture and mobility of the child adopting this position.CNS: Central nervous system; IMIP: Instituto de Medicina Integral Prof. Fernando Figueira; KM: Kangaroo Method; PTNB: Preterm Newborn; RMS: Root Mean Square; SENIAM: Surface Electromyography for the Non-Invasive Assessment of Muscles; SEMG: Surface Electromyography.The authors declare that they have no competing interests.The final submission of the manuscript was approved by all authors. KTD: Preparation of the study project, data collection and supervision thereof, statistical analysis, research articles in the database, preparation of the article. JECF: Guidance for the preparation of the study project, supervision of data collection, statistical analysis, research articles in the database; guidance and preparation of the article. RMM: Preparation of the study project, data collection and supervision thereof, data processing, statistical analysis, research articles in the database, preparation of the article. GMSL: Preparation of the study project, data collection and supervision thereof. DAV: Preparation of the study project, supervision of data collection, data processing.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2431/13/79/prepub


A frequent question from parents of adolescents is whether there are risk factors that could make a scoliosis curve increase, specifically whether carrying a heavy backpack on one shoulder could be detrimental to an existed curve. There has been very little research published to answer whether a unilateral upper extremity load would have an effect on spine position, let alone scoliosis progression .The purpose of this study was to measure changes in spinal curvature with increasing unilateral carrying load in order to analyze the possible risk for patients with adolescent scoliosis.Six young, non-scoliotic adults were measured with carrying loads of 15% and 20% of the subjects’ body weight compared to the neutral condition and analyzed for significant change. The formetric 3-dimensional/4-dimensional topography scanner was used to measure (1) weight distribution (2) kyphotic angle of the thoracic spine (3) lordotic angle of the lumbar spine (4) scoliosis angle of either the thoracic or lumbar spine, (5) coronal vertical axis (6) sagittal axis, and (7) shoulder tilt.Coronal imbalance had a small shift of about 4 mm off center towards the side holding the backpack. Sagittal imbalance shifted forward by about 15mm, regardless of the side holding the backpack. The pelvic obliquity changed by about 2° away from the side holding the backpack. Kyphosis and lordosis stayed fairly stable. Shoulder slope changed by about 8°, with the backpack side lower, resulting in a 2mm shoulder height difference. Weight shifted towards the side holding the backpack by about 20%, but did not shift towards the front or back.We found that coronal imbalance, sagittal imbalance, shoulder tilt and weight distribution changed significantly from the neutral position with 15% and 20% weight applied to either side of the body. Although it is not known what effect this would have on progression of scoliosis, these changes could potentially contribute in a negative way to spinal imbalance.


This publication is supported by the project 'BIOCEV - Biotechnology and Biomedicine Centre of the Academy of Sciences and Charles University - http://www.biocev.eu/en/' (CZ.1.05/1.1.00/02.0109), from the European Regional Development Fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." A grant number and the initial provider of the funding for the grant from BIOCEV were incorrectly omitted from the Funding Statement. The Funding Statement should read: "This work was supported by the Academy of Sciences of the Czech Republic grant no. IAA601110908 (


Congenital epulis is a very rare benign soft-tissue tumor of uncertain histogenesis, which is also known as “gingival granular cell tumor of the newborn”. It occurs almost exclusively as a single tumor along the alveolar ridge of the maxilla in newborn females. Although congenital epulis is strikingly similar to the more common adult granular cell tumor histologically, in contrast to the latter congenital epulis cells are negative for S-100 protein. This case report describes a 15-day-old female infant with multiple congenital epulis of the mandibular alveolar ridge. Congenital epulis is a rare benign soft-tissue tumor of uncertain histogenesis, which is also known as “gingival granular cell tumor of the new-born”. The first description of this lesion is attributed to Neumann in 1871-4.In this report, we describe the case of a 15-day-old female infant with multiple congenital epulis on her mandibular alveolar ridge and describe the clinical, pathological and differential diagnosis. The patient was a 15-day-old female infant born at 39 weeks gestation weighing 3200 g. She was born by normal vaginal delivery and had Apgar scores 9/10 at both 1 and 5 minutes. She was referred to our pediatric surgery department due to the presence of large intra-oral cavity masses that were protruding out of the oral cavity and causing feeding problems. These masses, first noticed at birth, originated from the anterior mandibular alveolus, occupied the entrance of oral cavity and partially protruded out of it.Intra-oral examination showed a reddish, pedunculated soft tissue mass exhibiting a smooth erythematous surface with some scales on the part outside the oral cavity. The mass measured 5 x 4 cm and was attached to the anterior gingiva of the mandible. Another two similar, smaller tumors were observed near the larger mass . On palpThe lesion was completely excised at the base with bipolar cautery under general anesthesia, with minimal intraoperative hemorrhage. Histologic examination of the specimens revealed well circumscribed lesions covered with squamous epithelium. They were composed of homogeneous polygonal cells with granular eosinophilic cytoplasm and centrally located basophilic nuclei in a vascularized stroma .(Periodic acid-Schiff ) positive. Immunohistochemically, the tumor cells were diffusely positive for vimentin, and negative for S-100 protein, actin and desmin. These findings were consistent with congenital epulis. No recurrence occurred during 3 years of follow up.The cytoplasmic granules were PAS Congenital epulis is a rare benign lesion of uncertain histogenesis. Although this tumor looks histologically like an acquired granular cell tumor , it has its own characteristics. Congenital epulis is a reactive or degenerative lesion with a mesenchymal origin rather than a true neoplasm . Recent Dash and colleagues reported on one of the largest series of congenital epulis to date, for which the authors collected data from 50 cases. Congenital epulis has an 8:1 incidence in females and a 3:1 incidence of occurring at maxillary alveolar sites . ConsideHistologically, congenital epulis is strikingly similar to the more common adult granular cell tumor. They are both composed of large round cells with abundant granular cytoplasm and small eccentric nuclei with occasional small nucleoli. There is a prominent vascular stroma with perivascular lymphocytes and histiocytes. Entrapped non-neoplastic odontogenic epithelium may be present in some cases. In contrast to granular cell tumors, there is no pseudoepitheliomatous hyperplasia of the overlying squamous mucosa in congenital epulis, and no nerve bundles are seen within this lesion. Furthermore, congenital epulis was shown to be consistently negative when immunostained with S-100 protein, in contrast to adult granular cell tumors, which are derived from Schwann cells ,4,5. SimCongenital epulis is often misdiagnosed before surgery because of its rarity and a lack of awareness of the condition by clinicians. The differential diagnosis of a large mass in the fetal or neonatal oral cavity includes hemangioma, lymphatic malformations, teratoma, pigmented neurectodermal tumor of infancy, rhabdomyoma and rhadomyosarcoma ,3,6. 3D Complete surgical excision is the treatment of choice but surgery should not be radical to minimize the danger of damaging underlying alveolar bone and developing tooth buds . There aCongenital epulis is a rare benign soft-tissue tumor of uncertain histogenesis. Cases of spontaneous regression, lack of recurrence even after incomplete resection and lack of malignant transformation suggest that congenital epulis is a non-neoplastic lesion and needs to be differentiated from an acquired granular cell tumor.


Dear Editor,9/L. Positron emission tomography/computed tomography showed multiple mildly hypermetabolic masses in the spleen and liver. Splenectomy and liver biopsy were performed. The spleen showed sinusoidal infiltration by large tumour cells with abundant foamy cytoplasm and active erythrophagocytosis. Tumour cells were positive for the histiocytic markers lysozyme, CD68 and CD163. Liver was also involved. Features were consistent with histiocytic sarcoma. Marrow examination showed the presence of large histiocytic cells with haemophagocytosis . There was a slow but relentless deterioration of her platelet count. Two years later, she was found to have splenomegaly and a platelet count of 23 × 10sis Fig. , which wsis Fig. . Haematozes Fig. . She wasThis patient initially presented with what appeared to be ITP. However, the gradual deterioration and the finding of a splenomegaly prompted further investigations , which s


Many factors influence breast cancer progression, including the ability of progenitor cells to sustain or increase net tumour cell numbers. Our aim was to define whether alterations in putative progenitor populations could predict clinicopathological factors of prognostic importance for cancer progression.Primary cultures were established from human breast tumour and adjacent non-tumour tissue. Putative progenitor cell populations were isolated based on co-expression or concomitant absence of the epithelial and myoepithelial markers EPCAM and CALLA respectively.Significant reductions in cellular senescence were observed in tumour versus non-tumour cultures, accompanied by a stepwise increase in proliferation:senescence ratios. A novel correlation between tumour aggressiveness and an imbalance of putative progenitor subpopulations was also observed. Specifically, an increased double-negative (DN) to double-positive (DP) ratio distinguished aggressive tumours of high grade, estrogen receptor-negativity or HER2-positivity. The DN:DP ratio was also higher in malignant MDA-MB-231 cells relative to non-tumourogenic MCF-10A cells. Ultrastructural analysis of the DN subpopulation in an invasive tumour culture revealed enrichment in lipofuscin bodies, markers of ageing or senescent cells.Our results suggest that an imbalance in tumour progenitor subpopulations imbalances the functional relationship between proliferation and senescence, creating a microenvironment favouring tumour progression. Significant interest surrounds the question whether cancer stem/progenitor cells drive tumour formation ,2, howevBreast progenitor cells are isolated based on expression of markers suggesting capabilities to generate cells of mixed myoepithelial and luminal epithelial lineages ,4. OtherUsing primary cultures from human breast tumour and non-tumour tissue, we sought to define correlations between progenitor cell numbers and clinicopathological or functional indicators of cancer aggressiveness. Our results demonstrate an imbalance between two putative progenitor cell populations in clinicopathologically-aggressive tumours, in conjunction with functional alterations promoting increased proliferation or reduced growth arrest. Taken together, full investigations of progenitor populations in relation to clinicopathological parameters could make an important contribution towards a better understanding of breast cancer progression.Suppliers: trypsin-EDTA, penicillin/streptomycin, penicillin/streptomycin/neomycin, fungizone, Cyquant, X-gal, Alexa-Fluor antibodies (Invitrogen); soybean trypsin inhibitor, collagenase I, hyaluronidase 1-S, DMEM/Ham's F12, bovine insulin, peroxidase-labelled secondary antibodies (Sigma); HMEC, mammary epithelial growth medium (MEGM) kits, foetal bovine serum ; glutaraldehyde (Fluka); osmium tetroxide (Electron Microscopy Services). Antibody suppliers: actin, ESA and SMA (Sigma); cytokeratin-19, PE-conjugated CALLA, FITC-conjugated EPCAM, FITC- or PE-conjugated IgG controls (Dako); cytokeratin-18 (Abcam); cytokeratin-14 (Millipore); vimentin and p63 (BD Biosciences).Breast primary cultures were generated from patient lumpectomy/mastectomy samples with informed consent as approved by the Medical Ethics committees of Beaumont Hospital and the Mater Misericordiae Hospital, in accordance with the Declaration of Helsinki. One piece each of tumour tissue and non-tumour margins  grown normally in DMEM-F12, 5% horse serum, 0.5 μg/ml hydrocortisone, 10 μg/ml insulin, 100 ng/ml cholera toxin, 20 ng/ml human recombinant EGF (MCF10A) or DMEM, 10% FBS, 2 mM L-glutamine(MDA-MB-231) were conditioned in MEGM for 2-3 weeks and used in flow cytometry experiments as controls for normal and tumourogenic phenotypes respectively.3) were plated in triplicate and harvested after 0, 3 or 6 days. Cyquant solution was incubated on freeze-thawed cells (5 min), and emitted fluorescence detected at 520 nm on a Wallac plate-reader. Fluorescence readings of unknown samples were translated into cell numbers by referring to two separate fluorescence standard curves - one for non-tumour and one for tumour cultures- constructed from known cell numbers  were plated in duplicate, and stained for senescence-associated β-galactosidase activity [Primary cells (5 × 10activity . Three bPrimary cells (passage 1-2) grown in chamber slides were fixed in 3.7% paraformaldehyde and immunostained for epithelial  or myoepithelial  markers using DAPI as a nuclear counter-stain. Primary antibodies were omitted in negative controls, and slides visualized on a Zeiss LSM510-meta confocal microscope.Confluent primary cultures were harvested in RIPA  containing protease and phosphatase inhibitors. Lysates were dounced and 25 μg supernatant subjected to SDS-PAGE and Western blot analysis for K19, K18, VIM and p63.+/EPCAM+, CALLA+/EPCAM-, CALLA-/EPCAM- or CALLA-/EPCAM+ populations on a BD FACSAria cell sorter. Some passage 0 cells were analyzed for activity of the stem cell marker ALDH by Aldefluor assay [5 cells were resuspended in assay buffer and incubated with activated substrate or the negative control reagent before analysis.Confluent passage 0 primary cells (T25 flask/condition) were trypsinized, blocked in human serum and co-incubated with FITC-conjugated mouse anti-human EPCAM and PE-conjugated mouse anti-human CALLA (4°C/30 min). Negative controls were unlabelled or single-stained with FITC-EPCAM, PE-CALLA, FITC-IgG or PE-IgG. Cells were analyzed on a Beckman Coulter Cyan-ADP and/or an Accuri-C6 flow cytometer. Cells were sorted into CALLAPassage 0 primary cultures or HMECs were fixed with 2.5% glutaraldehyde, processed as described  and analt-tests. Population variances were first compared using Instat-3.3.6 to inform the choice of equal/unequal variance between populations. The proliferation:senescence ratio was calculated based upon the data shown in Figure Data are expressed as mean ± standard error of the mean. Non-tumour versus tumour results were compared using non-parametric tests and one-tailed unpaired left). Two cellular populations were observed - large polygonal cells in colony centres , and small polygonal cells (spc) at the peripheries. Since spc and lpc resembled respectively myoepithelial and luminal epithelial cells, expression of epithelial and myoepithelial markers was examined by immunofluorescence microscopy  and tumour (T) human breast tissue yielded adherent organoids with outwardly-proliferating colonies Figure , left. Ty Figure . In compy Figure  detectioy Figure  also revNT versus VT). Multi-nucleation of tumour cells was frequently observed, in parallel with compromised nuclear membranes . Furthermore, tumour cell mitochondria were abnormal, elongated and occasionally fused . Finally, non-tumour cells displayed a well-differentiated rough endoplasmic reticulum (RER) while that in tumour cells was fragmented and dispersed .Ultrastructural analysis of matched cultures was undertaken to confirm differences between tumour and non-tumour specimens Figure . Firstlyleft). Since Cyquant proliferation assays quantify all cells rather than just actively-proliferating cells, we performed senescence-associated (SA) β-galactosidase assays [right). Non-tumour cultures had two-fold higher SA-β-galactosidase staining than that in tumour cultures. This was independent of the grade of the originating tumour, and did not reflect an impaired capacity to senesce in response to exogenous stimulation (data not shown).We next investigated if morphological differences were accompanied by cell fate differences Figure . Prolifee assays  to estimAs the balance between proliferation and senescence is more important than either parameter alone, we examined whether altered proliferation:senescence ratios in breast primary cultures could identify aggressive tumours. The proliferation:senescence relationship was estimated based on proliferation graph slopes and senescence values Figure . Our datarrow) resembling those described as progenitor/stem cells in mammary duct basal laminae [3b, top arrow). Layering of dark filament-rich cells  and EPCAM  markers ,12. All Given DN differences in aggressive HG or ER-negative tumours versus aggressive HER2-positive tumours, we performed ultrastructural analysis on DN populations from one non-tumour and one tumour culture . Although both populations had many similarities (data not shown), unique to the tumour DN population was the presence of abundant lipofuscin bodies Figure , arrows.Since both DN and DP populations are putative progenitor/stem cells ,4, we quin vivo.Intriguing recent work has suggested that immunohistochemical profiling of breast tumours for cancer stem cell populations may have prognostic value . To probMyoepithelial marker expression was found to dominate over luminal epithelial expression, consistent with observations in HMEC ,18. ExprMesenchymal/basal-like phenotypes also promote progenitor growth and tissue regeneration . The expWe sought to correlate subpopulation changes with tumour clinicopathological parameters, and observed decreased DP populations in aggressive tumours of high grade or ER negativity. ALDH activity was also reduced in HG tumours, an interesting fact since ALDH expression has been correlated with poor prognosis in breast cancer ,24 - altDN and DP populations have been described as slightly different putative progenitor/stem cell populations; with DN representing an undifferentiated population while DP represents a multipotent population ,12. SincInterestingly, we did not observe DN reductions in HER2-positive cultures. However elevated HER2 can drive premature senescence , and higWe have reported reduced senescence in tumour versus non-tumour breast primary cultures, and stepwise increases in the proliferation:senescence ratio with increasing tumour grade. Isolation of putative progenitor subpopulations revealed a novel correlation between increased DN:DP ratios and clinicopathological indicators of aggressive tumours . Our data suggest that progenitor population imbalance could promote tumour progression by altering the relationship between proliferation and senescence Figure . Future in situ; IDC: invasive ductal carcinoma; LC: lobular carcinoma; ITLC: invasive tubular lobular carcinoma; SA-β-gal: senescence-associated β-galactosidase; ER: estrogen receptor; PR: progesterone receptor; ESA: epithelial-specific antigen; SMA: smooth muscle actin; VIM: vimentin; CALLA: common acute lymphoblastic leukaemia antigen; EPCAM: epithelial cell adhesion molecule; DP: CALLA & EPCAM double-positive; DN: CALLA & EPCAM double-negative; HG: high grade; LG: low grade; ALDH: aldehyde dehydrogenase; TEM: transmission electron microscopy; K14: cytokeratin-14; K18: cytokeratin-18; K19: cytokeratin-19.MEGM: mammary epithelial growth medium; HMEC: human mammary epithelial cells; DCIS: ductal carcinoma The authors declare that they have no competing interests.SD and AMH conceived and designed the study, analyzed and interpreted the data, drafted the manuscript and revised it. SD performed most of the experimental work, with assistance from LH (primary culture generation), IA (senescence assay set-up), DCC (electron microscopy) and AB (cell sorting). DCC, AB and ADKH contributed to the interpretation of the results. ADKH, PAD, MJS, MS and MRK contributed to patient selection, sample acquisition and clinical interpretation. All authors read and approved the final manuscript.Primary culture patient information.Click here for fileProliferation assay standard curves for tumour and non-tumour cultures. Two non-tumour and two tumour cultures were used to generate standard curves to calculate numbers of cells from fluorescence values obtained at different time points of the Cyquant proliferation assays.Click here for fileMEGM medium does not alter the morphology of MCF-10A and MDA-MB-231 cells. MCF-10A and MDA-MB-231 cells were cultured for 15 days in MEGM or their standard serum-positive media, and imaged by phase contrast microscopy. No overt morphological differences were observed in either cell type after the media was switched.Click here for file


An 80-year-old Japanese male was diagnosed with pulmonary adenocarcinoma. The patient exhibited extensive extra pulmonary involvement in the bone, adrenal gland, abdominal lymph nodes, and sigmoid colon. A single course of chemotherapy with carboplatin and pemetrexed was administered as the first-line treatment. Subsequently, the patient received pemetrexed monotherapy. Two months after the diagnosis, rapid regression of the metastatic tumour in the sigmoid colon was observed. Based on the findings of CT scanning and colonoscopic examination, tumour-bowel fistulisation was considered to be a cause of the rapid regression. This case report illustrates a tumour-bowel fistula of a colonic metastatic tumour in a patient with lung cancer. Radiographic and endoscopic features of the rare manifestation are presented. Distant metastases are frequently encountered manifestations in patients with lung cancer. They can be present at the initial diagnosis or can occur in the treatment period. The bones, central nervous system, adrenal glands, and liver are representative organs involved in distant metastases –3. On thWe recently treated a patient with lung cancer who had extensive extra pulmonary involvement in the bone, adrenal grand, intra-abdominal lymph nodes, and sigmoid colon. Rapid regression of the metastatic tumour in the sigmoid colon was observed, and tumour-bowel fistulisation was considered to be a cause. Herein, we report the case and illustrate its radiographic and endoscopic features.An 80-year-old Japanese male underwent a chest x-ray examination at his family clinic due to the presence of dull pain around his shoulder. The patient was diagnosed with a lung tumour, and was referred to Onomichi Municipal Hospital for further investigation and treatment. He had a previous history of myocardial infarction and hypertension. He had a heavy smoking history of 80 cigarettes per day for 50 years. The patient had no history of pulmonary or gastrointestinal disease. Physical examination revealed no abnormalities. Laboratory studies revealed significantly elevated levels of carcino embryonic antigen  and sialyl stage-specific embryonic antigen-1 . Cytokeratin 19 fragment (CYFRA) concentration was also elevated (7.6 ng/mL). The levels of squamous cell carcinoma (SCC)-related antigen and gastrin discharge peptide precursor (ProGRP) were within the normal ranges.EGFR mutation status. The EML4-ALK fusion gene was also negative.CT scanning revealed a tumour in the left-upper lobe of the lung of 7.8 cm in diameter . The tumColonoscopic examination revealed a submucosal tumour in the sigmoid colon, which had ulceration on its top . Biopsy A single course of chemotherapy with carboplatin and pemetrexed was administered as the first-line treatment. Subsequently, the patient received pemetrexed monotherapy. Two months after the diagnosis, the patient underwent CT scanning because he exhibited a high fever. Inflammation around the kidney was detected by CT scanning, and pyuria was observed. Consequently, pyelonephritis was suspected as the cause of his fever. In addition, CT scanning showed the primary lung tumour and metastatic tumours to be unchanged, except that the sigmoid colon tumour had obviously regressed to 0.8 cm in diameter . The necApproximately, 50% of patients with lung cancer have distant metastasis at the initial diagnosis . Previouet al [N = 12), with fewer cases involving the stomach (N = 4) or large intestine (N = 2). Sakai et al noted that only 12 lung cancer cases with metastases to the large intestine have been reported in published case reports as of 2012 [N = 15, 88.2%). The pathological subtypes of the reported cases were SCC (N = 13), adenocarcinoma (N = 2) [N = 1), and large cell carcinoma (N = 1). The sigmoid colon is the most frequently metastasised site (N = 11), followed by the caecum (N = 3), ascending colon (N = 1), transverse colon (N = 1), and descending colon (N = 1). Therefore, a representative example of metastasis from lung cancer to the large intestine is a male patient with pulmonary SCC metastasising to the sigmoid colon.Within the gastrointestinal tract, the small intestine is the most frequently affected area, whereas colonic metastasis from lung cancer is extremely uncommon. Rossi et al , in a su of 2012 , 16–24.  of 2012 , 26. In  (N = 2) , small cTumour-bowel fistula is one of the potential complications of malignancies in the abdominopelvic cavity , 28. TheThe management of tumour-bowel fistula has not been established to date. Possible treatments for symptomatic patients include discontinuation of the preceding chemotherapeutic agents, administration of antibiotics, surgical resection of the tumour and fistula, and percutaneous drainage of the tumour abscess . We concWe experienced a rare case of primary lung cancer with metastasis to the sigmoid colon. The metastatic tumour rapidly regressed due to fistulisation into the bowel wall. Although such manifestations are infrequent, tumour-bowel fistula has to be suspected when a tumour close to the intestinal tract rapidly decreases in size.