Vitamin D plays an increasingly recognized role in the innate and adaptive immune response to infection. Based on demonstrated roles in up-regulating innate immunity, decreasing inflammation, and reducing the severity of disease in illnesses such as tuberculosis and influenza, we hypothesized that poor vitamin D status would be associated with severe malaria. We measured 25-hydroxyvitamin D [25(OH)D] by immunoassay in a sample of Ugandan children aged 18 months –12 years with severe malaria  and in healthy community children (n = 20). Ninety-five percent of children with severe malaria (n = 38) and 80% of control children (n = 16) were vitamin D-insufficient [plasma 25(OH)D <30 ng/mL]. Mean plasma 25(OH)D levels were significantly lower in children with severe malaria than in community children . Logistic regression revealed that for every 1 ng/mL increase in plasma 25(OH)D, the odds of having severe malaria declined by 9% [OR = 0.91 ]. These preliminary results suggest that vitamin D insufficiency may play a role in the development of severe malaria. Further prospective studies in larger cohorts are indicated to confirm the relationship of vitamin D levels to severity of malaria infection and to investigate causality. M. tuberculosis. Low plasma levels of vitamin D are associated with increased risk of active tuberculosis, while vitamin D supplementation accelerates recovery from the disease In addition to its established role in maintaining calcium homeostasis and optimal bone health, vitamin D plays an important immune-modulating role as a key player in innate and adaptive immunity 2D]. 1,25(OH)2D then binds to the VDR, increasing transcription of anti-microbial peptides (AMP), including cathelicidin, which results in improved response to anti-tuberculous therapy in individuals treated with vitamin D Work by Liu et al. 2D 2D, have decreased inflammation and improved survival when exposed to influenza A as compared to untreated In addition to helping clear infection, vitamin D can also lessen disease severity by reducing inflammatory cytokines produced as by-products of immune activation. To date, this anti-inflammatory action of vitamin D and resulting improved clinical outcomes have been most clearly demonstrated with influenza, with a recent clinical trial reporting a decrease in pulmonary inflammatory response to influenza in individuals treated with 1,25[OH]Plasmodium falciparum also includes activation of the innate immune system via toll-like receptors on macrophages in vitroA potential beneficial role of vitamin D in malaria infection has not yet been well described. However, as with tuberculosis, the immune response to Plasmodium berghei malaria, those that received oral supplementation with vitamin D did not develop experimental cerebral malaria Supporting a beneficial anti-inflammatory role of vitamin D in malaria, a recent study reported that among mice infected with To determine whether vitamin D status was associated with severe malaria in children, we determined 25(OH)D levels and the prevalence of vitamin D insufficiency in Ugandan children aged 18 month –12 years with severe malaria and in healthy community controls selected from the same household or village who were within one year of age. We hypothesized that the children with severe malaria would have lower levels of 25(OH)D, based on the evidence cited above that vitamin D may strengthen the innate immune response and protect against severe infection through decreased inflammation. A finding that vitamin D deficiency is more prevalent among children with severe malaria than among community children would suggest a potential role for vitamin D deficiency in the etiology of severe malaria and could lay the groundwork for future studies testing vitamin D supplementation as an adjuvant therapy for the condition.From 2008–2013, we conducted a study of the pathogenesis of severe malaria and its neurocognitive outcomes at Mulago Hospital, Kampala, Uganda. Children with severe malaria [cerebral malaria (CM) or severe malarial anemia (SMA)] and healthy community children (CC) who were between 18 months and 12 years of age were enrolled and followed for two years to document neurobehavioral development.The present analysis includes plasma samples collected at baseline from a subsample of 60 children [40 children with severe malaria  and 20 CC] enrolled in the larger study. These samples for the present analysis were randomly selected from the larger sample using a computer-generated algorithm in FileMaker Pro .P. falciparum on blood smear; and 3) no other known cause of coma. SMA was defined as P. falciparum on blood smear plus hemoglobin level ≤50 g/L. CC were within one year of age of children with severe malaria and were recruited from the same family or neighborhoods as children with CM or SMA. Eligibility criteria for CC were: 1) age 18 months to 12 years; 2) currently healthy; and 3) no illness requiring medical care within the previous four weeks. Known chronic illness was an exclusion criterion for all study groups.For the parent study, CM was defined as: 1) coma (Blantyre Coma Score [BCS] ≤2 or Glasgow coma score [GSC]≤8); 2) A venous blood sample of 5–7 mL was drawn into a heparinized blood collection tube from each child. Blood was centrifuged, and plasma was collected and stored at −80°C.Plasma concentrations of 25(OH)D were measured by chemiluminescent immunoassay at the University of Minnesota Fairview Diagnostics Laboratory, Minneapolis, MN, a CLIA-certified laboratory. Vitamin D insufficiency was defined as a 25(OH)D level <30 ng/mL Written informed consent was obtained from parents or guardians of all study participants. Ethical approval was granted by the Institutional Review Boards for human studies at Makerere University School of Medicine, University of Minnesota, and Michigan State University.We compared baseline socioeconomic, demographic, and clinical characteristics of CC and children with severe malaria using a t-test for normally distributed variables and Wilcoxon-rank sum test for non-Gaussian distributions. Proportions were tested using chi-square or Fisher’s Exact test when cell sizes were small (<5 children).In unadjusted analysis, we first used a t-test to compare mean 25(OH)D in children with severe malaria to CC. We used ANOVA to test for differences in 25(OH)D level among the 3 groups, i.e., CM, SMA, and CC, and applied Tukey’s test to account for multiple comparisons. We then adjusted for potential confounders between malaria risk and vitamin D deficiency, including nutritional status , age, and sex, using multiple logistic regression with severe malaria as the outcome variable and 25(OH)D, anthropometric indices, age, and sex as predictor variables.Community children and children with severe malaria were similar with regard to age, sex, height-for-age z-score, and indicators of socioeconomic status and food security Table 1.Plasma concentrations of 25(OH)D were low overall and were significantly lower among children with severe malaria than among community children . This relationship did not change after controlling for weight-for-age z-score  or weight-for-height z-score .In the present study, we found that vitamin D insufficiency is widespread among young Ugandan children and that 25(OH)D levels are significantly lower among children with severe malaria than otherwise healthy community children. To our knowledge, this is the first study to investigate vitamin D status in the context of malaria in children. The finding of an association between low 25(OH)D and severe malaria suggests a possible role of vitamin D deficiency in the etiology of severe malaria and establishes the foundation for larger studies. Confirmation of the findings of the present study would set the stage for testing vitamin D supplementation as a preventative or adjunctive therapeutic intervention for severe malaria.The study finding that 80% of healthy community control children were vitamin D insufficient was surprising, particularly among a group of physically active children living near the equator and having year-round exposure to UVB radiation. Although the religious affiliation of the children in our study is not known, Uganda is 85% Christian and having children fully covered is uncommon. A recent study in Malawian infants and toddlers While vitamin D levels were low overall, children with severe malaria have significantly lower 25(OH)D levels than community children. This finding was surprising and cannot be explained by differences in socioeconomic status or food security. To date, only one study has evaluated vitamin D levels over the course of a malaria infection A recent study proposed that vitamin D binding protein must be considered when assessing vitamin D status M. tuberculosis, and to level of inflammatory response to malaria.Limitations to our study include the small sample size and the cross-sectional design, which limits our ability to make any conclusions regarding a causal effect of 25(OH)D level on severity of malaria. The study findings should be viewed as preliminary data, but the finding that 25(OH)D was significantly lower among children with severe malaria than among community children merits further investigation. Future studies should assess vitamin D levels in a larger cohort of children, and test whether vitamin D levels in these children relate to production of anti-microbial peptides, such as cathelicidin, via a mechanism analogous to that observed with In conclusion, we found that vitamin D insufficiency is common in Ugandan children, that children with severe malaria have significantly lower levels of 25(OH)D than healthy community children, and that lower levels of vitamin D are associated with increased odds of severe malaria. Future studies investigating a possible role for vitamin D in mounting an optimal immune response to malaria and in protecting from severe disease are warranted.Data S1(XLSX)Click here for additional data file.


Cystic fibrosis is one of the most frequent inherited rare diseases, caused by mutations in the cystic fibrosis transmembrane conductance regulator gene. Apart from symptomatic treatments, therapeutic protocols for curing the disease have not yet been established. The regeneration of genetically corrected, disease-free epithelia in cystic fibrosis patients is envisioned by designing a stem cell/genetic therapy in which patient-derived pluripotent stem cells are genetically corrected, from which target tissues are derived. In this framework, we present an efficient method for seamless correction of pF508del mutation in patient-specific induced pluripotent stem cells by gene edited homologous recombination. Gene edition has been performed by transcription activator-like effector nucleases and a homologous recombination donor vector which contains a PiggyBac transposon-based double selectable marker cassette.This new method has been designed to partially avoid xenobiotics from the culture system, improve cell culture efficiency and genome stability by using a robust culture system method, and optimize timings. Overall, once the pluripotent cells have been amplified for the first nucleofection, the procedure can be completed in 69 days, and can be easily adapted to edit and change any gene of interest.The online version of this article (doi:10.1186/s13287-016-0275-6) contains supplementary material, which is available to authorized users. Genetic correction by homologous recombination (HR) had proven extremely inefficient in human embryonic stem (hES) or human induced pluripotent stem (hIPS) cells before the advent of genome editing  and the Gene edition with recombinant nucleases follows a multi-step procedure mainly divided into three sections: 1) creating specific double strand breaks (DSBs) by engineered nucleases, near to the desired locus on the genome; 2) triggering the homology-directed repair (HDR) process and editing the gene by means of adding a donor vector with homology arms and selectable markers as a repair template for homologous recombination; and 3) seamlessly removing selectable markers from isolated clonal or semiclonal populations. Repair template contains homology arms matching 5’ and 3’ regions surrounding the DSB point. Selectable markers are needed to identify successfully corrected genomes. The rationale behind this repair has been highly argued and readers are referred to the literature for a detailed explanation .At present, there are three major kind of engineered nucleases: zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats/associated (CRISPr/Cas) nucleases . All theElimination of the selectable markers after genetic repair with most common Cre/loxP, Flp/FRT, and ϕC31 integrase/attP-attB recombination systems leave small ectopic sequences in the genome, which even if located in intronic regions could have the potential to interfere with transcriptional regulatory elements , splicinpuroΔtk) in a transposon in the repair vector was developed previously and applied to the correction of A1AT deficiency in patient-specific iPS cells [The addition of transposons/transposase as an alternative to the list of recombinase mechanisms  has consPS cells . After hPS cells .Cystic fibrosis (CF) is one of the most frequent rare diseases, caused by mutations in the CFTR gene. Apart from symptomatic treatments, there is no alternative to cure the disease at present . RecentlTk expression was conducted by treatment with fialuridine (1-(2-deoxy-2-fluoro-1-D-arabinofuranosyl)-5-iodouracil; FIAU), an analog of ganciclovir. Surviving clones were expanded and checked by PCR for elimination of the selectable cassette, and CFTR alleles were cloned and sequenced on several of them to identify successfully repaired patient-specific CFTRpMC3.1/pF508Δ. One hIPS cell line from each of two patients was corrected. CF is a monogenic recessive disease, therefore heterocygotic correction is sufficient to restore CFTR function in repaired cells.In the present work, we designed and constructed a PiggyBac-based HR vector, and nucleofected it in combination with TALENs specifically designed to cut in the region near the CTT (pF508del) deletion of the human CFTR gene. Positive selection of the recombinant clones was performed by resistance to puromycin, and polymerase chain reactions (PCRs) designed to detect the integration of vector homology arms were conducted on genomic DNA from all of them. Hyperactive transposase was transfected in a list of more than ten positively targeted clones, and upon transposon removal a negative selection for There are seven major stages in the correction of disease-associated genetic mutations by TALEN-edited seamless HR in patient-specific pluripotent cells: 1) the generation of patient-specific iPS cells including confirmation of the mutation and its homo- or heterocygosis; 2) the design and construction of the nuclease and the donor vectors for HR; 3) the repair experiment itself with selected nucleases according to their cutting efficiency and the preparation of replicated cultures of positively selected populations; 4) the design and carrying out of the PCR screening to assess positive candidates; 5) the elimination by transposition of the selectable double marker in chosen candidate populations and negative selection procedure; 6) the cell cloning and screening of selected populations for the effective elimination of the selection cassette; and 7) the confirmation of the bona fide correction and the absence of other unwanted genetic modifications in chosen repaired clones.Patient-specific hIPS cell line establishment and characterization has been performed according to the work already described elsewhere , 25. In http://zifit.partners.org). CompoZr® Knockout ZFN, engineered ZFNs for pF508Δ targeting from Sigma™, were supplied under a collaboration agreement. TALEN vectors designed to cut in the same spot as ZFNs were designed and purchased from Transposagen®.ZFN expression vectors were originally designed using ZiFiT targeter software provided by the Zinc Finger Consortium  create a restriction site (for BglII as it has unique cutting site in the whole recombination region) which will be useful in the identification of the recombined alleles during screening; b) avoid recognition of the recombined allele for the nucleases (re-targeting); and c) design specific primers for the recombined allele versus the endogenous ones. Besides the homology arms, the synthetic (control) vector designated as Seq_1 contained homology arms surrounded at 5’ and 3’ ends by specific genomic regions chosen as primers for PCR detection of recombination events, respectively. EcoRI and MluI sites were designed at both ends of each string, respectively, so they could be easily eliminated from the control vector during construction of the targeting vector.http://www.sanger.ac.uk/form/-C3ZIws9wQ4CSysnb1AY4kA) under an MTA agreement.Construction of the donor vector for HR experiments was thus performed in three steps of modification of this synthetic control vector: first, EcoRI digestion of Seq_1 eliminated the contiguous genomic region in the 5’ arm (generating Seq_2); then MluI digestion eliminated the contiguous genomic region in the 3’ arm (Seq_3); in the third step, digestion of the NheI/NotI fragment from the pMCS-AAT-PB:PGKpuroΔtk vector was cloned into NheI/NotI sites in Seq_3, introducing the PuroΔtk/PiggyBac (PB) transposon cassette in between the homology arms to generate the donor targeting vector pMC3.1 (Seq_4). PMCS-AAT-PB: PGKpuroΔtk is the targeting vector used for A1AT correction  and was The pMC3.1 targeting vector, besides having a strong double selection cassette, allows for screening strategies for each step of the HR procedure: PCR amplification for positive recombinant clones, restriction fragment analysis of the recombinant allele to check the stability of its open reading frame (ORF), and detection of the reverted allele after excision of the transposon/selection cassette, when we can use the point mutations located at the nuclease recognition sites to amplify specifically recombined and non-recombined (either mutated or corrected) alleles. This screening system shares a common forward primer and uses two different reverse primers which will recognize native from recombined alleles.CelI assay , 27 for hIPS cell lines were generated and described elsewhere . Brieflytk cassette. Briefly, subconfluent cells were trypsinized and resuspended in HESNEW medium containing ROCK inhibitor before centrifugation at 200 g and resuspension in nucleofector solution 2 . B16 program was used for best results combining nucleofection efficiency and cell viability. Nucleofected cells were plated onto resistant feeders described before in the presence of ROCK inhibitor for 1 day in HESNEW_S, and thereafter the media was substituted by the puromycin-containing HESNEW_R at 1 μg/ml. Emerging resistant clones were allowed to grow and subcultured individually up to 16 days after plating, feeding them every second day. Grown colonies were manually picked and transferred to 48 tissue culture microplates, which were grown for an additional 5 to 8 days. Clones were then replicated by trypsinization, and grown until at least 50 % confluence. One replica plate was frozen and the other was used for genomic DNA extraction which was performed manually by a basic protocol. Briefly, cells were lysed in high salt content buffer and genomic DNA was precipitated with isopropanol and washed in ethanol before resuspension in Tris-EDTA buffer.CFhIPS3.18 and CFhIPS4.4 were nucleofected with ZFN and pMC3.1 plasmids at a ratio of 1 μg of each ZFN and 2 μg of the targeting vector per million cells. For TALEN experiments, TALENs and donor vector were mixed and nucleofected at a ratio of 2 μg of each circular vector per million cells using an Amaxa nucleofector device II and following manufacturer instructions. After nucleofection, hIPS cells were seeded onto HFF-1/W3R feeders , kindly Genomic DNA from 48-well replica plates of colonies resistant to puromycin was extracted by proteinase K digestion of the cells on the plate by lysis buffer and precipitation of the DNA with isopropanol after transferring lysates to eppendorf tubes. DNA pellets were washed in 70 % ethanol and resuspended in Tris-EDTA buffer. PCRs were performed at annealing temperatures according to temperature gradient optimization for each pair of primers, and extension times according to product length , and then Reint primers were used to confirm that no random reintegration of the transposon occurred in the genome.Tk negative events resulting from the elimination of the selectable cassette, resistant colonies were isolated and duplicated in P48 well plates. One replica was expanded in culture while the other was used for PCR screening and DNA sequencing. CFTR alleles were cloned and sequenced in both senses with 5’ and 3’ primers separately, and reactions were purified with a column and sent to Secugen®. Results were visualized in Chromas software and browsed in GenBank for identity check.Confirmed positive clones for HR were thawed and expanded, and subjected to cassette excision by nucleofection of 5 ug of the pCMV-HAhyPBase vector that encodes the transposase per million cells. FIAU selection was applied from day 1 onwards after nucleofection, and resistant clones were mechanically split and subcultured for at least three passages until genomic DNA was extracted for analysis. After FIAU selection for Stage 7 of DNA repair, which is confirmation that no other mutations have been generated during the repair procedure, was completed by PROGNOS software and Sanger sequencing of the predicted off-target sites. Predicted Report Of Genome-wide Nuclease Off-target Sites (PROGNOS) online software from Gang Bao laboratory was used to evaluate potential rearrangements in off-target sequences throughout the genome after pF508Δ correction by HR . Target The same genomic DNA from corrected lines was submitted to comparative genome hybridization array (aCGH) analysis (outsourced).A panel of ten hIPS lines was established from keratinocytes and fibroblasts of two CF patients and one healthy donor (in preparation). Stable pluripotent lines were continuously cultured and expanded to prepare frozen stocks at low passages 6–15). Additional file 5. AdditiBasic human pluripotent culture is best routinely maintained in parental HFF-1 fibroblasts which are irradiated at 40 Gy and frozen as batches of around 180 million cells each in vials of 2–3 million cells each. Growth and amplification of resistant populations is then best achieved using resistant human fibroblasts, which are robust feeders during the relatively long period of culture for clonal growth of rare events, as positive recombinants. With this system, feeder supplementation is avoided at any time and pluripotent culture is robustly supported.Efficiency of CompoZr® CFTR_ZFN was calculated to be 12.4 % by Sigma in the K562 human promyelocytic leukemia cell line. Surveyor assay for TALENs resulted in a cutting efficiency of 2.76 % in one of our CFhIPS pluripotent cell line  PB selection cassette, offers three different screening strategies. The first one is a PCR amplification of each recombined homology arm that only will be positive once the recombination is achieved. In these PCRs, clones that are positive for 5´ and 3´ hybrid sequences were detected. The second screening implies the digestion of the 5’ arm with Bgl-II and their sequencing, in order to check the stability of the CFTR ORF. The third screening step was performed after cassette excision, where silent mutations introduced to avoid nuclease retargeting after correction were used to design a three-primer PCR system that will amplify distinctively the non-recombined alleles (wild-type (wt) used as a PCR-positive control and pF508Δ) from the recombined allele. This screening system shared a common forward primer and was based in two different reverse primers that recognized and amplified the native CFTR/CFTRpF508Δ (wt/mutated) allele or the recombinant CFTR/pMC3.1 (wt/edited) allele.In previous HR strategies, the removal of the selection cassettes left residual sequences in the edited locus, which although placed more often in the intron part of the edited gene implied locus restrictions for HR strategies. The new strategy using transposon/transposase as the recombination method avoids the introduction of foreign sequences in the recombinant allele. Furthermore, the pMC3.1 vector, apart from having a strong + (puro)/–(Δ−5, resulting in a feasible protocol in terms of both workload and timing. Raw data corresponding to the homologous recombination experiments was communicated at a symposium organized by the Spanish Society of Gene and Cell Therapy, and is available online at http://www.setgyc.es/home/ZFN_3_M_Camarasa.pdf.The use of TALEN-mediated HR in combination with a transposon-based double selectable marker achieved correction of pF508Δ mutation in hIPS lines from two patients at efficiencies ranging 1.7-4.9 x 10Development of generations of hIPS cells towards clinical production have been reviewed . The pre


NCAM2 and TSHR and decreased expression of GCNT1 was associated with decreased PFS and OS after IV chemotherapy (p < 0.05). High tumor expression of LMAN2, FZD4, FZD5, or STT3A was associated with no significant PFS increase after IP compared to IV chemotherapy. Low expression of APC2 and high expression of FUT9 was associated with 5.5 and 7.2 months, respectively, decreased OS after IP compared to IV chemotherapy (p ≤ 0.007).Tumor mRNA expression was used to discover genes associated with worse survival or no survival benefit after intraperitoneal (IP) chemotherapy. Data for high grade serous ovarian cancer patients treated with IP (n = 90) or IV-only (n = 398) chemotherapy was obtained from The Cancer Genome Atlas. Progression free survival (PFS) and overall survival (OS) were compared between IP and IV groups using Kaplan-Meier analysis and Cox regression. Validations were performed by analyses of microarray and RNA-Seq mRNA expression data. PFS and OS were compared between IP and IV groups by permutation testing stratified by gene expression. P-values are two-tailed. IP chemotherapy increased PFS , p = 0.0001) and OS , p = 0.01). Increased expression of  Ovarian cancer is the deadliest gynecologic malignancy, with 44.6% five-year overall survival (OS) and 1% lifetime mortality risk34BRCA1 protein expression measured by immunohistochemistry of primary tumor specimens was associated with 36 months increased OS among patients treated with IP compared to IV-only adjuvant chemotherapyDespite available strategies for preemptive management of side effects of IP chemotherapy, widespread adoption of IP chemotherapy remains limited by toxicities8Chemotherapy information was from the TCGA drug dataset131215We used the R platform for statistical computing18Within each adjuvant chemotherapy group, cases were stratified by PFS time <or ≥12 months. Twelve months was used because cancers that recurred or progressed before 12 months were likely chemoresistant. Patients who undergo surgical staging and six cycles of adjuvant chemotherapy often reach 6 months after completion of chemotherapy, the time at which platinum sensitivity is designated, about 12 months after surgery. A diagram of the exploratory gene analysis and validation steps is shown in 14RMS curves were plotted for differentially expressed genes as functions of normalized relative microarray mRNA expression21KM survival curves comparing survival by chemotherapy route are shown in ASPA, overall survival) indicates that the risk of death increased 30% for each 1 standard deviation increase in microarray mRNA expression across the range of observed expression values. Since expression data varies over several standard deviations for each gene, survival effects for some genes are large comparing the lower and upper limits of gene expression . Almost half  of the differentially expressed genes were associated with PFS and/or OS by multivariate regression using microarray data  (NCAM2 and TSHR and decreased expression of GCNT1 was associated with decreased PFS and OS after IV chemotherapy (p < 0.05)   and 4. H step 1) . Only 5/ step 1)  and 3. I < 0.05) .APC2 or high expression of BCAT1 experienced less survival benefit from the addition of IP chemotherapy to their treatment  of FZD5 showed an expected overall survival benefit among the IP chemotherapy group but did not show a significant PFS benefit (no significant difference between IP and IV-only groups) .Since patients treated with IP chemotherapy typically have significantly increased OS and PFS compared to patients who receive IV-only chemotherapy, biomarker evaluation for response to IP chemotherapy may aim to discover primary tumor biomarkers that are associated with worse or no better survival after IP chemotherapy. RMS curves demonstrate PFS and OS by relative gene mRNA expression (Supplementary RMS curves) allowing for evaluation of survival as a function of gene expression across the range of observed gene expression values. Pointwise 95% confidence intervals around each univariate RMS curve allow visual comparison of OS and PFS between IP and IV groups (Supplementary RMS curves). Illustrative RMS curves are shown . Comparireatment . Patientotherapy . Table 4Adjuvant IP chemotherapy is associated with increased PFS and OS in TCGA data. A large and increasing number of publications reported survival outcomes of TCGA ovarian cancer patients without adjusting for chemotherapy route as a potential confounder. Reanalysis of previously reported survival outcomes to adjust for chemotherapy route as a potential confounder may be needed given that IP chemotherapy use was common among TCGA HGS OvCa patients and that receiving IP chemotherapy was associated with large and highly significant increases in PFS and OS.APC2, FZD4, and FZD5, which had mRNA levels that were significantly associated with OS and PFS, are members of the Wnt signaling pathway. The observation that increased expression of APC2 and decreased expression of FZD4 and FZD5 was associated with decreased OS and PFS among patients treated with IV chemotherapy suggests that downregulation of Wnt signaling may play a role in chemoresistance2425A detailed review of the tumor biology of all candidate biomarkers is beyond the scope of this report. DAVID gene functional classification failed to reveal any clusters among differentially expressed genesLimitations of our study include incomplete clinical data reporting to TCGA, a relatively small number of patients who received IP chemotherapy, and also small numbers of tumors that were analyzed by RNA-Seq, especially among the IP group. During the RNA-Seq validation, many survival associations trended (p = 0.05–0.10) but failed to reach significance due to few cases having RNA-Seq data. Clinical and chemotherapy information reported to TCGA was also not verified for correctness. We could not perform validation in another patient cohort because no other suitable data is available. To our knowledge, TCGA provides the only cohort of ovarian cancer patients treated with IP chemotherapy for which there is currently transcriptome or proteome data. Also, tumor protein expression was not confirmed experimentally by immunohistochemistry of primary tumor specimens. The TCGA reverse phase protein array does not include any of the differentially expressed genes and therefore the TCGA proteomics data could not be used as a potential protein-level validation of our transcription-level findings. In addition, the process of gene discovery by t-testing and gene validation with limited RNA-Seq data is very stringent. Thus, there are likely additional genes that are significantly associated with survival outcomes among this patient cohort. Targeted rather than exploratory analyses of well-known molecular pathways driving cancer development or chemotherapy resistance may lead to discovery of additional genes with similar survival associations after adjusting for chemotherapy exposure.Strengths of our study include use of a standard and fully reproducible statistical test (the t-test) to compare gene expression levels between groups. We also analyzed gene mRNA expression levels as a continuous variable in all regression models of PFS or OS. Dichotomizing or otherwise arbitrarily subdividing cases by gene expression thresholds is very commonly reported in the literature but is less informative than RMS analysis. RMS curves illustrate the relationships of gene expression and survival. We provide TCGA case identifiers with chemotherapy route to ease reproduction of our findings and reanalysis of previous studies.in vivo models. A targeted clinical assay for efficient measurement of primary tumor gene expression of these candidate biomarkers may be developed. Some gene products may be amenable to measurement in serum or ascites, or by primary tumor immunohistochemistry. Our findings may be corroborated at the level of protein expression by performing immunohistochemical analysis of primary tumor specimens, if made available, from a previous randomized trial of IP versus IV chemotherapy34Adjuvant IP chemotherapy of TCGA patients was associated with increased PFS and OS similar to randomized trials34How to cite this article: Seagle, B.-L. L. et al. Discovery of candidate tumor biomarkers for treatment with intraperitoneal chemotherapy for ovarian cancer. Sci. Rep.6, 21591; doi: 10.1038/srep21591 (2016).


Osteoarthritis (OA) is the fastest growing cause of disability worldwide. In the A number of dietary factors have been associated with OA symptoms or progression. Most notably, in those overweight, weight reduction of ≥10% has the potential to lead to important changes in pain and function. Losing It has been suggested that OA is a metabolic disease in which lipids essentially contribute to the pathophysiology of cartilage degradation. DietaryA positive association has been shown between elevated serum cholesterol and OA; hypercholesterolemia  and high serum cholesterol  were independently associated with generalized OA in the Ulm study[Vitamin D affects the state of multiple articular structures. The evidence for association between the vitamin D biomarker, serum 25(OH)D, and OA was assessed in a systematic review. For knee radiographic OA progression and cartilage loss, there was strong evidence for an association with low 25(OH)D.Vitamin K is important in cartilage metabolism as an inhibitor of extracellular matrix calcification and a promoter of cell survival/proliferation. In the US MOST study, vitamin K deficiency was associated with incident radiographic knee OA and MRI-based cartilage lesions  compared with no deficiency[Hence, dietary recommendations are the following:▪ lose weight, if overweight, preferably combined with exercise;▪ reduce plasma cholesterol by dietary means;▪ at least for a trial period, increase intake of long-chain n-3 fatty acids preferably by eating oily fish twice a week;▪ aim for a safe level of sun exposure, eat rich vitamin-D dietary sources or take vitamin D supplements, ≤ 25 µg/d;▪ increase vitamin K intake by eating green leafy vegetables.


Hepsin,  and TMPRSS3 are type II transmembrane serine proteases (TTSPs) that are involved in cancer progression. TTSPs can remodel extracellular matrix (ECM) and, when dysregulated, promote tumor progression and metastasis by inducing defects in basement membrane and ECM molecules. This study investigated whether the gene and protein expression levels of these TTSPs were associated with breast cancer characteristics or survival.TMPRSS1 mRNA expression was determined in 125 invasive and 16 benign breast tumor samples, and TMPRSS3 mRNA expression was determined in 167 invasive and 23 benign breast tumor samples. The gene and protein expression levels were analyzed for associations with breast cancer-specific survival and clinicopathological parameters.Immunohistochemical staining was used to evaluate hepsin levels in 372 breast cancer samples and TMPRSS3 levels in 373 samples. TMPRSS1 and TMPRSS3 mRNA expression levels were independent prognostic factors for poor breast cancer survival during the 20-year follow-up . Low expression of the two genes at the mRNA and protein levels associated with poorer survival compared to high levels . Low TMPRSS1 mRNA expression was also an independent marker of poor breast cancer prognosis in patients treated with radiotherapy . Grade III tumors, large tumor size, and metastasis were associated with low mRNA and protein expression levels.Low The results suggest that the TTSPs hepsin and TMPRSS3 may have similar biological functions in the molecular pathology of breast cancer. Low mRNA and protein expression levels of the studied TTSPs were prognostic markers of poor survival in breast cancer.The online version of this article (doi:10.1186/s12885-015-1440-5) contains supplementary material, which is available to authorized users. TMPRSS1 gene, and TMPRSS3, encoded by the TMPRSS3 gene.Globally, breast cancer is the most commonly diagnosed cancer in women, while metastatic disease is the leading cause of cancer-related deaths in this group . EpithelTMPRSS1 mRNA overexpression is associated with ER(α)-positive human breast tumors   . BrilliaFor immunohistochemical staining, 372 invasive breast cancer tumor samples were available for hepsin analysis and 373 samples were available for TMPRSS3 analysis. Immunohistochemical staining was performed on 4-μm sections cut from the tissue microarray (TMA) blocks. The TMA blocks were constructed with a custom-built instrument . The sample diameter of the tissue core in the array block was 1000 μm and three samples from tumor tissue of each case were studied. After deparaffinization and rehydration, the sections for TMPRSS3 analysis were heated in a microwave oven for 3 × 5 min in citrate buffer (pH 6.0). The sections for hepsin analysis were not heated. The slides were treated for 5 min with 5 % hydrogen peroxide to block endogenous peroxidase, then incubated for 35 min at room temperature in 1.5 % normal serum diluted in PBS to block non-specific binding. The blocked sections were incubated overnight at 4-°C with the rabbit polyclonal primary antibody against hepsin  at a dilution of 1:250 or with an antibody against TMPRSS3  at a dilution of 1:250. The slides were then incubated with a biotinylated secondary antibody for 35 min and with an avidin-biotin-peroxidase complex for 45 min . Slides were rinsed with PBS after each step of the immunostaining procedure. The color was developed using diaminobenzidine tetrahydrochloride . The slides were counterstained with Mayer's hematoxylin, washed, dehydrated, cleared, and mounted with Depex . For the negative controls, the primary antibody was omitted.Three slides from each TMA block were examined in triplicate by two researchers  under the supervision of a senior pathologist (YS). The immunoreactivity of hepsin and TMPRSS3 in the cytoplasm of epithelial tumor cells was analyzed, and the intensity and the extent of staining were scored . The non-parametric Mann-Whitney U test and the Kruskal-Wallis test were used to study differences in continuous mRNA expression values according to different clinicopathological parameters. Fisher’s exact test was used to study associations between protein expression and clinicopathological parameters. The odds ratios (ORs) and the 95 % confidence intervals (95 % CIs) were determined using logistic regression analysis to describe the strength of statistically significant associations between expression levels and clinicopathological characteristics. The Kaplan-Meier method was used in univariate survival analyses. Multivariate Cox’s proportional hazards analysis was carried out in a forward stepwise method to estimate the hazard factors having an impact on breast cancer-specific death and relapse. In addition to mRNA and protein expressions, Cox regression analysis examined the following clinicopathological parameters: tumor grade, nodal status, tumor size, estrogen receptor (ER) status, progesterone receptor (PR) status, and tumor histologic type. In addition, the adjuvant treatments were used as variables in the analyses including the treatment data. All statistical tests were two-sided, and a TMPRSS1 and TMPRSS3 as prognostic biomarkers in breast cancer  [P values are calculated [We used an online Kaplan-Meier survival analysis tool to validate the value of =breast) . The Kaplculated .TMPRSS1 and TMPRSS3 mRNA expression, and Additional file TMPRSS1 and TMPRSS3 mRNA expression was high in well-differentiated malignant breast tumors compared to benign breast tumors  and that low hepsin expression was positively associated with larger tumor sizes , which indicates more extensive disease. The Mann-Whitney U test showed that low TMPRSS1 and TMPRSS3 expression levels were associated with ER-negative status, PR-negative status, and HER2-positive status .In the multivariate Cox regression survival analysis, low mRNA expression of TMPRSS3 mRNA and low levels of TMPRSS3 protein had more frequent relapses  and poor overall survival .Associations between the studied expression levels and breast cancer-specific and overall survival according to the adjuvant therapies given to the patients were analyzed using univariate Kaplan-Meier analysis and multivariate Cox regression survival analysis. Low TMPRSS3 mRNA expression was associated with poor breast cancer-specific survival  in all the treated patients and remained an independent factor predicting more frequent relapse occurrence . Low TMPRSS3 protein expression also predicted poorer relapse-free survival  compared with high TMPRSS3 expression. No significant results were found between different treatment groups regarding TMPRSS3 expression.When the treatment data was included in the survival analyses, low TMPRSS1 expression levels correlated with hepsin protein expression levels , and TMPRSS3 expression levels correlated with TMPRSS3 protein expression levels . Positive correlations were also found between TMPRSS1 and TMPRSS3 mRNA expression  and between hepsin and TMPRSS3 protein expression . Consequently, combined TMPRSS1 and TMPRSS3 mRNA expression and combined hepsin and TMPRSS3 protein expression were tested for statistical association with clinicopathological parameters and breast cancer-specific survival. The combination variables were formed so that the breast cancer cases that expressed low mRNA levels of both of the studied genes formed the ‘low combined mRNA expression group’ and the remaining cases formed the other (reference) group. A similar approach was used to forming a ‘low protein expression’ group. Breast cancer cases with low combined expression were associated with clinicopathological parameters that indicate advanced tumor malignancy , low mRNA expression remained an independent factor for breast cancer survival. Similarly to our results, Dhanasekaran et al. showed previously that low hepsin protein expression in human prostate cancer samples correlated with poor prostate cancer prognosis . In theie cancer . Roemer survival . They susurvival . In humaval time . These sTMPRSS1 and TMPRSS3 expressions are associated with the occurrence of relapses and that low TMPRSS3 mRNA and protein expression are independent factors affecting distant metastasis occurrence. Aberrant expression of TTSPs is associated with tumor invasion and metastasis in various epithelial cancers [TMPRSS3 mRNA and protein expression remained independent factors that affected relapse in the Cox regression analyses. On the other hand, based on prostate cancer cell line studies, Srikantan et al. suggested that hepsin overexpression could have antitumorigenic effects and hinted that hepsin might be involved in some sort of positive feedback response [In this study we have shown that altered  cancers , 41. Sup cancers . This hyresponse . They suresponse . Wittig-response . Taken tBased on their proteolytic activity at the cell surface, TTSPs could contribute to tumor progression by affecting initiation of the metastatic process in primary breast cancer tumors. Several substrates for hepsin have been linked to epithelial carcinogenesis, including HGF and uPA. Hepsin and another TTSP, matriptase, efficiently convert inactive pro-HGF to biologically active HGF that, in turn, activates the HGF receptor c-Met , 22. ThiTMPRSS3 expression in cancer has not nearly been studied as extensively as the expression of TMPRSS1. More work needs to be done to study the biological role of TMPRSS3 in cancer. Nonetheless, our study presents in a coherent clinical breast cancer sample set that TMPRSS3 is a credible prognostic biomarker. In contrast to our results, a previous study presented that hepsin overexpression was associated with positive nodal status and tumor stage in breast cancer [TMPRSS1 and TMPRSS3. Based on the survival curves displayed and the logrank P values both low TMPRSS1 and TMPRSS3 expression significantly associated with poorer relapse-free survival, overall survival, and distant metastasis-free survival [The limitations of our study include that t cancer . Howevert cancer . Similarsurvival . To sum TMPRSS1 mRNA expression remained an independent factor of poor prognosis in patients who were treated with radiotherapy. It must be highlighted that low TMPRSS1 expression remained the only significant variable regarding prognosis which excludes for example poor differentiation level of breast cancer cells in these analyses. Furthermore, no significant results were found in patients who were not given any adjuvant therapies. When TMPRSS1 expression level is higher and epithelial integrity is still rather intact it might be that radiation induced cellular lethality is much more aggressive in breast cancer cells. However, when TMPRSS1 expression is low and epithelial integrity already damaged it appears that the remaining breast cancer cells are radioresistant leading to these cells surviving which has negative impact on the clinical outcome. Our results indicate that low TMPRSS1 expression may independently reduce the therapeutic function of radiation yet the specific cellular mechanisms remain unclear. Interestingly, Nakamura et al. showed in an endometrial cancer cell line study that hepsin overexpression resulted in significant cell accumulation at the G2/M phase leading to cell cycle arrest [TMPRSS1 and TMPRSS3 SNP genotypes that associated with survival in patients treated with radiotherapy [When the treatment data was included in the multivariate survival analyses, low e arrest . Cancer e arrest . These potherapy . We haveST14 gene) is a TTSP that, similar to hepsin, can activate pro-HGF. A recent study showed that hepsin and matriptase are direct pericellular activators of pro-HGF and hypothesized that their suggested ability to autoactivate might be the initial step in HGF/c-Met-mediated basement membrane degradation [The combinations of low expression levels of mRNA and protein were independent factors that predicted poor survival. This suggests that TTSPs are prognostic biomarkers for breast cancer. Matriptase (encoded by the radation . Our preradation . Howeverradation . NotablyTMPRSS1 mRNA expression is an independent marker of poor clinical outcome in patients treated with radiotherapy. We think that our results give emphasis to the role of altered expression of hepsin and TMPRSS3 in promoting breast tumor progression and metastasis as their role in breast cancer is still rather unexplored. The results showed that both TTSPs have potential as prognostic biomarkers.In closing, this study expands our knowledge of the biological processes behind breast cancer by investigating hepsin and TMPRSS3 expression in human breast tumors. Low mRNA and protein expression levels of the studied TTSPs were prognostic markers of poor survival in breast cancer. Furthermore, low


Germline mutations in the phosphatase PTEN are associated with diverse human pathologies, including tumour susceptibility, developmental abnormalities and autism, but any genotype-phenotype relationships are poorly understood.We have studied the functional consequences of seven PTEN mutations identified in patients diagnosed with autism and macrocephaly and five mutations from severe tumour bearing sufferers of PTEN hamartoma tumour syndrome (PHTS).All seven autism-associated PTEN mutants investigated retained the ability to suppress cellular AKT signalling, although five were highly unstable. Observed effects on AKT also correlated with the ability to suppress soma size and the length and density of dendritic spines in primary neurons. Conversely, all five PTEN mutations from severe cases of PHTS appeared to directly and strongly disrupt the ability to inhibit AKT signalling.Our work implies that alleles causing incomplete loss of PTEN function are more commonly linked to autism than to severe PHTS cases. Phosphatase and tensin homologue deleted on chromosome ten (PTEN) is a phosphatase that suppresses the activity of the class I phosphoinositide 3-kinase/AKT signalling pathway.9–11The major diagnostic criteria for PHTS include malignancies of the breast, thyroid and endometrium in addition to benign hamartomas, skin lesions and macrocephaly.supplementary figure S1A, B). This apparent difference between assay formats encouraged us to analyse a larger group of seven mutant proteins identified in patients with autism (supplementary figure S3A). These data show that the mutations identified in patients with autism do not greatly reduce the ability of the expressed PTEN proteins to inhibit cellular AKT signalling and imply that these mutants retain catalytic activity.Recombinant PTEN protein purified from bacteria has been used in many previous studies to demonstrate that phosphatase activity is critical for its tumour suppressor function. In preliminary experiments studying three PTEN mutants identified in patients with autism, we could detect little or no catalytic activity in vitro from protein purified from bacteria, yet transient expression of these mutants in mammalian cells led to a robust suppression of the phosphorylation of the PTEN regulated kinase AKT (see online h autism A lackingh autism C. Invest3) vesicles. Recovered activities were weak for many of the PTEN mutants, but this correlated with a low recovery of immunoprecipitated PTEN, probably due to poor stability of these mutants during the immunopurification process (see online supplementary figure S2). In particular, PTEN H118P, F241S, D252G and N276S were recovered poorly and gave very weak activity. Activity normalised to reflect PTEN protein content showed robust activity for all mutants, although weaker than wild type enzyme (see online supplementary figure S2). These experiments also indicate that testing the protein phosphatase activity would be problematic. In addition to this difficulty immunoprecipitating active protein, during these experiments, it was noted that to express most of the autism-related PTEN missense mutations at the same level as PTEN WT protein, a higher titre of lentivirus was consistently required (see online supplementary figures S2A and S3A). Therefore, we investigated the possibility that these mutations affected the stability of the PTEN protein.To test their phosphatase activity directly, we overexpressed each protein in U87MG cells and assayed freshly immunopurified PTEN proteins in vitro against radiolabelled phosphatidylinositol 3,4,5-trisphosphate  the amount of protein expressed was very low D, E.To assess more directly the stability of these mutant proteins relative to PTEN WT, we used the protein synthesis inhibitor cycloheximide (200 µg/mL). Transduced cells were incubated with the inhibitor for 0 h, 2 h, 4 h, 6 h and 8 h, followed by immunodetection using anti-PTEN antibody A, B. WhePten is deleted to investigate whether the physiological level re-expression of PTEN WT or autism-associated PTEN mutants is able to support a wild type phenotype of normal dendritic growth and neuronal size. Hippocampal neurons prepared from E15.5 flox/flox Pten mice were transduced simultaneously with lentiviruses expressing Cre recombinase (RFP-Cre) to delete endogenous Pten and with viruses encoding PTEN WT or PTEN mutants. The mutants PTEN H123Q, F241S and D326N were selected to represent a range of protein stabilities (see Pten genes and the re-expression of human PTEN mutants were verified by western blot (Pten had not been deleted), relative to neurons expressing Cre, with or without additional inactive PTEN C124S. In agreement with the previous results, we found that the autism-related mutants H123Q and D326N were capable of controlling these morphological changes in broad correlation with the amount of protein expressed, showing effect similar to PTEN WT when expressing similar amount of protein. PTEN F241S expressed very poorly and did not affect neuronal signalling or morphology in accordance with its very low apparent stability.Macrocephaly is one of the several anatomical and cellular abnormalities that have been suggested to be important factors in the pathogenesis of ASD.ties see . The delern blot D. Deletiern blot B, C of hern blot E–G to lePTEN mutations, we chose to select a comparator group of mutations associated with a severe tumour bearing PHTS phenotype. To do this, we conducted an extensive search of data published in journals and online resources associating specific PTEN mutations with phenotype (see online supplementary tables S1–S3). Although inconsistencies in the categorisation of patients’ severity are likely to be introduced by diversity in the diagnosis and recording of separate phenotypical characteristics between publications, our analysis identified 112 missense mutations that had sufficient associated phenotypical data and which had been identified in a total of 204 patients . This analysis shows that missense mutations (rather than truncating mutations) were overrepresented among all PTEN mutation carriers described with ASD compared with all carriers of PTEN mutations, consistent with the hypothesis that more of these mutants may retain some activity (figure S3).Considering the view that patients diagnosed only with ASD and macrocephaly may reflect a group at the milder end of the spectrum of phenotypes associated with supplementary figure S3B). Analysis of the half-life of PTEN mutant proteins showed that two of the mutations (L108P and L112P) destabilise the protein with a decrease in protein levels by ∼60–80% after 8 h of protein synthesis inhibition, whereas the protein level for the mutants A39P, N48K and R130L, as well as for PTEN WT, C124S, G129E and G129R was reduced by ∼20% (Five PTEN missense mutations associated with the most severe phenotype (score >6) were selected for characterisation . These by ∼20% D, E. ThiOur data show that all seven PTEN mutant proteins that we investigated due to their identification in patients with ASD retained the capacity to suppress cellular phosphoinositide 3-kinase-AKT signalling when they were expressed to the same level as wild type PTEN. However, most of these mutant PTEN proteins are unstable and it would be expected that in vivo this instability would lead to a very considerable reduction in protein abundance relative to PTEN WT and a resultant reduction in biological activity. Functional evaluation of PTEN mutants in a yeast based system has previously found significantly lower activity in a set of PHTS/Cowden-associated PTEN mutations than in autism-associated mutations,In contrast, when a set of PTEN mutations associated with the most severe cases of Cowden syndrome were tested for their cellular activity, all of these five mutant proteins were found to display no detectable effect on AKT phosphorylation even when overexpressed. This implies that complete loss of activity may associate with a more severe PHTS/Cowden phenotype. Of these five proteins, three showed stability similar to wild type PTEN, whereas two were unstable. In the cases of the stable mutants, this supports a hypothesis that the expression of stable inactive protein may exacerbate the phenotype caused by PTEN loss of function, through interference with the functions of the remaining WT PTEN protein and possibly other proteins. Strong evidence supporting the potential importance of this effect comes from the observed phenotypes of Pten knockin mice, in which the most severe tumour phenotype was observed in mice expressing stable PTEN mutants lacking lipid phosphatase activity, G129E or C124S, relative to either mice carrying a Pten deletion or with an unstable PTEN mutation.Our work implies that mutation-specific factors do contribute to the severity of PTEN mutation carriers’ symptoms. These effects may relate to the retention of some biological PTEN activity in the case of patients displaying macrocephaly and ASD in the absence of more severe developmental phenotypes, and to the expression of dominant negative inactive PTEN protein in some cases combining severe developmental and eventually tumour phenotypes. Biochemically, potential dominant negative effects of mutant PTEN proteins have not been extensively studied, although data supporting the functional importance of PTEN dimerisation has very recently been published.flox-flox murine neurons, a modified lentiviral vector was used in which the expression of the RFP-Cre transgene is driven by a human synapsin-1 promoter.U87MG glioblastoma cells culture, lentivirus preparation and titration were as previously described.flox-flox mice (E15.5) and digested in trypsin to obtain dissociated cells as described in Kreis et al.2 for immunocytochemistry and biochemical analysis.Hippocampi were isolated from embryonic day 15.5 Pten RFP-Cre lentivirus particles were added to the neuronal culture 7 days after seeding, together with lentivirus particles encoding WT or mutant forms of PTEN and left for 6 days. Every 2–3 days half of the medium was replaced by fresh medium. Neuronal cells used for immunocytochemistry were transfected with yellow fluorescent protein 24 h before fixation by using Lipofectamine 2000 as transfection reagent (0.6 µg DNA/0.8 µL Lipofectamine in Optimem). Of the medium 250 µL was removed, collected in a tube, mixed with fresh media in a 1:1 ratio and incubated at 37°C. Transfection mix was added to the cells for 20 min and then removed and replaced by the incubated media. Cells used for biochemical analysis were washed in phosphate buffered saline (PBS) and lysed using RIPA buffer , protease inhibitors and phosphatase inhibitors).


Mycobacterium bovis bacille Calmette–Guérin (BCG) vaccines, given to neonates to protect against adult tuberculosis and the risk of using these live vaccines in HIV-infected infants, has emphasized the need for generating new, more efficacious and safer replacement vaccines. With the availability of genetic techniques for constructing recombinant BCG (rBCG) strains containing well-defined gene deletions or insertions, new vaccine candidates are under evaluation at both the preclinical and clinical stages of development. Since most BCG vaccines in use today were evaluated in clinical trials decades ago and are produced by outdated processes, the development of new BCG vaccines offers a number of advantages that include a modern well-defined manufacturing process along with state-of-the-art evaluation of safety and efficacy in target populations. We provide a description of the preclinical development of two novel rBCGs, VPM1002 that was constructed by adding a modified hly gene coding for the protein listeriolysin O (LLO) from Listeria monocytogenes and AERAS-422, which carries a modified pfoA gene coding for the protein perfringolysin O (PFO) from Clostridium perfringens, and three genes from Mycobacterium tuberculosis. Novel approaches like these should be helpful in generating stable and effective rBCG vaccine candidates that can be better characterized than traditional BCG vaccines. The failure of current  Although still one of the most widely used vaccines in the world, BCG originally developed by Calmette and Guérin more than 90 years ago and first administered to infants in 1921 . Two of hly from Listeria monocytogenes coding for the protein LLO, and inactivation of BCG’s gene for urease subunit C (ureC) to allow the acidification of the phagosomal compartment. Vakzine Projekt Management, GmbH (VPM) has facilitated the further development and acts as a sponsor for the clinical testing of this novel rBCG vaccine. The rBCG vaccine, VPM1002 was originally developed by Stefan H.E. Kaufmann and his team at the Max Planck Institute for Infection Biology in Berlin ,5. Two gMycobacterium tuberculosis (Mtb) antigen (Ag85B) in BCG Tice. This vaccine was shown to demonstrate substantially better protection vs. Mtb challenge in an animal model compared to its parental BCG strain, and elicited Ag85B specific CD4 T cell responses in humans [pfoA gene coding for the protein perfringolysin O (PFO) from Clostridium perfringens, with a mode of action similar to listeriolysin [The non-profit product development organization, Aeras, has sponsored the development of two rBCGs that have entered clinical trials. The first rBCG vaccine, rBCG30, overexpressed a single n humans . Althougn humans , it was ntigen Ag5B in BCGIn this review, we provide the details on the developmental strategies used to design and characterize these two novel rBCG vaccines and in addition, summarize the methods and tests used to manufacture and evaluate the candidate products to meet regulatory requirements and describe the pre-clinical assays developed to characterize the critical parameters of these live TB vaccine candidates. Although pharmacopeial guidelines exist for the testing and evaluation of traditional BCG vaccines , differeureC locus by insertion of a hly gene coding for an LLO expression cassette. The leader sequence of Ag85B was included in front of LLO to allow for secretion of the protein [ureC gene were introduced upstream and downstream of the hygromycin B resistance cassette of the mycobacterial recombination vector. The LLO expression cassette was then inserted between the 3'-region of ureC and the antibiotic marker to result in the final construct, which was electroporated into the parental strain to allow for homologous recombination. Individual hygromycin resistant clones were selected from Middlebrook 7H11 agar containing 80 µg/mL hygromycin B. Genetic modifications were confirmed by PCR, Southern blotting and deep sequencing.VPM1002 was constructed by a single step that disrupted the  protein . For con600, contents were harvested, cross filtered, and the concentrated bulk was combined with formulation buffer. The material was then filled into 2 mL amber serum vials and lyophilized, stoppered, crimp-sealed, labeled and stored at 2–8 °C and used for the following nonclinical studies. VPM1002 was adapted to animal-free Sauton’s medium to create a Master Seed Bank (MSB) and used to produce the Working Seed Bank (WSB). Bulk Drug Substance (BDS) was produced using three 200 mL cultures grown in shake flasks containing Sauton’s medium. The contents of the flasks were used to inoculate a 30-L fermenter containing Sauton’s medium. After meeting the desired growth OD6 viable bacilli of BCG Danish-SSI 1331, VPM1002 or saline. After 90 days mice were infected with a low dose (200 CFUs) of Mtb H37Rv per animal using a Glas-Col inhalation exposure system. At 30, 60 and 90 days post-challenge, six mice per group were sacrificed, and lungs and spleens homogenized in saline with 0.05% Tween 80 and plated in serial dilutions onto Middlebrook 7H11 agar. Plates were incubated for 3–4 weeks at 37 °C prior to enumerating viable bacterial colony forming units (CFU). Experiments had ethical clearance by the Landesamt fuer Gesundheit und Soziales Berlin under permit number G0307/11.Female BALB/c mice were vaccinated subcutaneously with 1 × 105 CFU and a total of 6 female and 6 male guinea pigs per experiment were randomly allocated to two treatment groups of 6 animals each . Body weight was determined three times a week during the observation period, animals were checked three times a week for clinical findings and mortality was recorded daily. At the end of the observation period all animals were necropsied and macroscopically altered organs were evaluated histopathologically.The purpose of these studies was to obtain information on the toxicity of VPM1002 in male and female guinea pigs after single subcutaneous application of the test item followed by periods of 6 to 26 weeks and dosages up to 50-fold human target dose (HTD). The HTD was considered 1–4 × 105) of VPM1002, BCG Danish-SSI 1331 or saline followed by an observation period of 90 days, with samples collected 10, 21 and 90 days post-vaccination for gross necropsy and histopathology. Special attention was paid to the local tolerance at the injection site. Target organs were investigated for their mycobacterial load by evaluating the number of CFUs in tissue samples. VPM1002 colonization in target organs was stained and assessed by microscopy. The body weight of each newborn rabbit was monitored on the day of administration, twice weekly until the end of week 2, and weekly from week 3 onwardsInformation derived from this study serves to indicate test-item-related local intolerance reactions, signs of systemic toxicity, mortality and mycobacterial load in target organs. Newborn rabbits were vaccinated subcutaneously within the first 2 days after colostrum feed with either one human target dose  was ligated with the 2.7 kb antigen cassette generated by PCR from pRC102. pRC131 was digested with PacI to remove the antibiotic marker and self-ligated. The self-ligated, purified plasmid was electroporated into AERAS-413  and used to produce the Master Cell Bank (MCB). The Bulk Drug Substance (BDS) was produced using three MCB vials grown in shake flasks containing 7H9 medium. The contents of the flasks were used to inoculate a 20-L fermenter containing 7H9 medium. After meeting the desired growth of ODvs. FSC-H to exclude doublets. CD3 T cells were then gated against V450 , followed by gating on CD4 and CD8 T cells. Female Chinese rhesus macaques, between 2–5 years of age, were used in this study. Animals were housed at Advanced Biosciences Laboratory, Inc , in accordance with the standards of the American Association for Accreditation of Laboratory Animal Care. Animals were randomized into groups including saline (n = 4), BCG Danish-SSI 1331 (n = 6) and AERAS-422 (n = 6) and were immunized intradermally with 100 µL of saline, BCG Vaccine SSI (1 × 106 CFUs) or AERAS-422 (1 × 106 CFUs). Animals were bled at weeks 0, 6, 8 and 12 and PBMCs were isolated by standard Ficoll-Hypaque method  with LIV6 CFUs of BCG Danish-SSI 1331 or AERAS-422 as described previously [In a mouse protection study approved by the institutional animal care and use committee of the Center for Biologics Evaluation and Research, C57BL/6 mice were vaccinated subcutaneously with saline, 5 × 10eviously . After 86) of BCG Danish-SSI 1331, AERAS-422 and AERAS-401 . Mice were sacrificed when moribund; defined by severe lethargy, hunched with ruffled fur, loose skin or other signs of severe distress in line with FELASA recommendation on limiting clinical signs.Six- to 8-week-old female SCID mice  were immunized with 100 µL (5 × 105 CFUs/0.1 mL adult dose). To test excessive dermal reactivity in line with European pharmacopeia and WHO recommendations [4) or AERAS-422 (5 × 106) and observed for adverse effects. Testing for freedom from virulent mycobacteria was performed to detect the presence of virulent mycobacteria in the lyophilized vials of AERAS-422 to be used in human clinical trials. Guinea pigs were injected subcutaneously with two doses of AERAS-422, low (2.5 × 107) and high (1 × 108), to achieve a target dose of 10 and 50 times the highest anticipated clinical dose. General safety of AERAS-422 was evaluated in both mice and guinea pigs with a five times higher dose  and a 50 times higher dose  than the highest anticipated human dose.For repeat dose toxicity study, guinea pigs were injected intradermally with approximately 10 times greater than the current recommended BCG dose given to humans . Urease catalyzes the hydrolysis of urea into carbon dioxide and ammonia, thus creating a basic environment. Inactivation of this gene is necessary since listeriolysin is optimally active under acidic conditions, as previously mentioned. Both modifications were engineered in one approach by the insertion of hly into the ureC locus, thus disrupting the urease sequence and deleting its activity [M. bovis BCG ∆ureC::hly .The rBCG currently in the most advanced clinical studies is VPM1002. VPM licensed the vaccine candidate from the Max Planck Society and has sponsored the preclinical-clinical development to the current clinical phase IIa studies in neonates. This novel construct was produced by integrating the e of BCG . LLO is e of BCG ; (2) LLOe of BCG ,18,19. Ie of BCG ,5. MHC Il escape , and is l escape . The tarVPM1002’s preclinical proof of concept for efficacy and safety has been analyzed in 10 studies comprising approximately 600 animals. In general, all studies were performed using BCG Danish, sub type Prague vaccine as a baseline for vaccine induced responses. For efficacy, mice were vaccinated with VPM1002 or BCG and subsequently challenged by aerosol, using either the laboratory strain Mtb H37Rv or a clinical isolate of Mtb. Efficacy was determined by enumerating viable Mtb colony forming units (CFUs) in the lung and spleen. Preclinical testing of VPM1002 in mice demonstrated better protection from Mtb infection compared to BCG SSI . The immThe safety of VPM1002 was evaluated in a number of animal models and in three different species. Interferon-gamma (IFNγ) knock-out mice served as a model for evaluating the risk of disseminated infection with BCG and VPM1002 vaccines in severely immunocompromised humans, since BCG infection has been related to deficiencies in the IFNγ signaling pathway. No VPM1002-vaccinated animal died during the observation period of 105 days in contrast to BCG , and no adverse effects on animal health were observed. However, both VPM1002 and BCG induced local reactions at the site of vaccination.et al., 2005 [i.e., 5 × 106 CFU per animal), the severity and incidence of findings were similar or less severe than the single human dose of the reference BCG strain (given at 5 × 105 CFU). Bridging studies with cGMP-manufactured vaccine showed comparable levels of safety in SCID mice.The target product profile for clinical development of VPM1002 considered HIV-infected individuals, therefore further safety studies were performed in SCID mice which lack an adaptive immune system. As described in Grode Three single-dose toxicity studies were performed in guinea pigs with follow-up periods of 6 to 26 weeks and dosages up to 50-fold human target dose (HTD). In general, weight gain (a highly sensitive marker for TB in guinea pigs) was similar in all treatment groups and in the range of normal variation known for this species. No guinea pig died prior to study end and no lesions typical for TB or related mycobacterial infections were observed during necropsy or histopathological evaluation. Small white spots (approx. 1 mm) were seen on the livers of some animals from all groups . Histopathology of those spots revealed cell necrosis and hydropic degeneration in both BCG and VPM1002. The pathology seen by microscopy and the naked eye following vaccination with VPM1002 was less than that seen with BCG and no viable bacilli were detected in lung, liver or lymph nodes as observed by culture, although PCR revealed a similar systemic spread of both VPM1002 and BCG nucleic acid in the above mentioned organs. In a safety model using new born rabbits and comparing VPM1002 to BCG (both human target/standard dose), no mortality was observed. Macroscopically and histologically, all animals showed clear infectious reactions, which are expected at the 2-week time point post-immunization with a live BCG vaccine. A preclinical study in newborn rabbits was also performed to compare VPM1002 with BCG for safety, including bio distribution of the bacteria 90 days post-vaccination. The body weight of male and female animals vaccinated with BCG (control) did not gain the same amount of weight compared to animals treated with 0.9% saline. No influence on body weight was noted for the animals treated with VPM1002 . Also thpanCD genes coding for pantothenate synthase were deleted [panCD complementation, which helped to retain the episomal plasmid containing two classical Mtb antigens (Ag85A and Ag85B) and one latency-associated antigen (Rv3407), without the need for an antibiotic marker. The overexpression of these antigens by this new rBCG strain, AERAS-422, was confirmed by Western blot analysis using specific antibodies to the Mtb antigens. Antigen expression was also stable after infecting cells from the human macrophage-like cell line, THP1, and plating at different time points and checking colonies for the presence of genes coding for the above mentioned antigens using PCR  modified to express PFO, AERAS-401, has been described previously . Using ssing PCR . Both NHPs ,24 and mp < 0.05) post-challenge and in spleens at both 12 and 20 weeks (p < 0.05) post-Mtb challenge  or BCG in lungs 12 weeks  of mice immunized subcutaneously with BCG SSI died within the 300 day observation period, while 9 out of 10 mice immunized with AERAS-422 survived as did the naïve control group . As mentAERAS-422 was produced as cGMP compliant material using qualified manufacturing procedures. For toxicological evaluation and release of the cGMP material, acute toxicity, repeat dose toxicity, skin test reactivity (PPD) and freedom from virulent mycobacteria studies were performed. Guinea pigs were used for all of these studies due to their sensitivity to TB ,27. In rThe World Health Organization (WHO) Expert Committee on Biological Standardization has recently revised the “Recommendations to Assure the Quality, Safety and Efficacy of BCG Vaccines”, which is used by global manufacturers of BCG vaccines . Althougvs. challenge experiments, which are commonly the most important factor in deciding whether to move a vaccine candidate forward, investigators have used various BCGs as comparators and various immunization and Mtb strains and challenge strategies [As previously described , investirategies . As showrategies , rBCGs crategies  that timnew well-characterized products manufactured by state-of-the-art technologiessafe and effective in contemporary clinical trials compared to currently used traditional BCG vaccinesshown to be may serve as excellent prime vaccines for novel booster TB vaccines currently under development.A new rBCG vaccine may be more acceptable (marketable) to the global community, as long as it can be shown to be safer and more efficacious than BCG, and if the cost is comparable or lower than the currently available BCG. Unlike traditional BCG vaccines, novel vaccines offer several advantages including:A robust process for developing candidate products that have undergone extensive preclinical studies such as those described in this article would facilitate acceptance with regulatory agencies for clinical development. In general, it would provide a better-defined product than BCG licensed decades ago by traditional methods. Current BCG vaccines can be relatively reactive vaccines that cause ulceration, scarring and lymphadenopathy, as well as serious adverse effects from dissemination in immunocompromised infants ,40,41. RA strategy presently employed by sponsors of TB vaccine candidates in clinical trials is the use of TB subunit vaccines to boost subjects previously vaccinated with BCG. However, several of these antigens are expressed by BCG at low levels or not at all . DevelopIn this report, we have provided examples of testing strategies that have been successfully applied to two new rBCG vaccines that have entered human clinical trials . Althoug


Laquinimod is a novel orally administered drug for the treatment of relapsing remitting multiple sclerosis (RRMS). In this immunological substudy of the phase III Assessment of Oral Laquinimod in Preventing Progression of MS (ALLEGRO) trial, we performed an ex vivo and in vitro analysis of effects exerted by laquinimod on peripheral blood immune cell populations from RRMS patients with a special focus on monocyte phenotype and function. Approximately 100 patients were enrolled following a standardized protocol. Half of the patients received laquinimod and the other half received placebo. Peripheral blood samples were collected prior to commencement of therapy and after 1, 3, 6, 12, and 24 months of continuous therapy. Main lymphocytic and antigen presenting cell fractions were analyzed in peripheral blood mononuclear cells (PBMCs) ex vivo by flow cytometry. The proliferative response of PBMCs to mitogen or recall antigen was assessed in culture experiments. Untouched monocytes were sorted magnetically and cultured under pro-inflammatory conditions. PBMC analysis showed no significant differences of investigated lymphocytic and antigen presenting cell populations over time within each group, or between the two groups. However, the detailed in vitro analysis of monocytes demonstrated a lower level of CD86 expression on monocytes stimulated with LPS in laquinimod patients beginning from the 1st month of treatment. Upon pro-inflammatory stimulation, monocytes obtained from laquinimod treated patients tended to secrete lower levels of the proinflammatory chemokines CCL2 or CCL5. Taken together, in this prospective study, we demonstrate immune modulation but no immunosuppressive biological activity of laquinimod in a large group of MS patients. It was selected based on an extensive structure-activity relationship program, which showed a high oral bioavailability, increased potency and a superior safety profile versus other candidates In inflammatory and autoimmune models other than EAE, laquinimod reduced or prevented clinical manifestations in an experimental model of systemic lupus erythematosus In MS patients, laquinimod was effective in suppressing MRI-measured disease activity in both phase IIa and IIb clinical trials, and was well tolerated without any opportunistic infection In this immunological substudy of the phase III ALLEGRO trial, we sought to evaluate the immunological effects of laquinimod on PBMC populations and monocyte phenotype/function in RRMS patients. We focused on patients enrolled in Germany and analyzed a panel of cellular and humoral immune functions.Prospective longitudinal analysis of changes in PBMC populations and monocyte phenotype and function was performed in patients enrolled in the phase III ALLEGRO trial. Eighty-seven patients gave informed consent to participate in this immunological substudy and were enrolled from multiple sites across Germany (12 ALLEGRO study centers participating in the substudy). Forty-four patients received laquinimod and 43 patients received placebo. There were no significant differences in the main demographic and clinical parameters between the groups of this substudy. For further patients characteristics see TableVenous blood (20 mL) was drawn into EDTA-containing tubes at individual study sites. Upon receipt of blood samples, peripheral blood mononuclear cells (PBMC) were isolated from anti-coagulated blood by centrifugation on a discontinuous density gradient . The mononuclear cell fraction was washed three times in phosphate buffered saline (PBS), counted and suspended in PBS for flow cytometric analysis and isolation of untouched monocytes.+ monocytes , and 10% heat-inactivated fetal calf serum . For pro-inflammatory stimulation experiments, culture medium was supplemented with 100 U/mL of recombinant interferon-gamma  or 1 µg/mL lipopolysaccharide . After 48 h of culture cells were harvested for flow cytometric analyses and culture supernatants were collected for measurement of inflammatory cytokine and chemokine production.In order to obtain untouched monocytes for in vitro experiments, PBMCs were negatively sorted with the Untouched Monocyte Isolation Kit II  in the magnetic field of a MidiMACS® sorter (Miltenyi Biotec), to deplete lymphocytes and granulocytes. The resulting negative fractions typically contained >90% of CD14onocytes . Isolate+, CD3+CD4+, CD3+CD8+), B cells (CD19+), NK cells (CD56+CD3−), NKT cells , monocytes (CD14+), mature dendritic cells (CD83+). Regulatory T cell populations were identified as CD4+CD25highFoxP3+ lymphocytes. Intracellular staining for Foxp3 was performed on freshly isolated PBMC with the PerCP Anti-Human Foxp3 Staining Set  according to manufacturer's protocol. Before fixation, the cells were counterstained with mAbs specific for CD4 and CD25.PBMCs were assessed by multicolor flow cytometry using a FACSCanto II® cytometer and FACSDiva® software . The major mononuclear cell populations were recognized by staining with fluorochrome conjugated monoclonal antibodies (mAb) specific for key surface antigens: T cells . All data were expressed as percent of the whole PBMC fraction.5 PBMC with or without stimuli in triplicates. After 48 h of culture at 37°C, 5% CO2, wells were pulsed with 0.2 µCi 3H-thymidine for 18 h; then cells were harvested and 3H incorporation was determined by scintillation counting. Data were calculated as stimulation index, the x-fold increase in cpm incorporation with stimulus as compared to background cpm. Absolute antigen or mitogen-specific responses were reported for each sample as well as the change from baseline.The relative proliferation of PBMC in the presence of phytohemagglutinin (PHA) or tetanus toxin antigen was assessed. The response of PBMC to mitogen (PHA—5 µg/mL) or recall antigen  was assessed by plating 10Monocyte culture supernatants were analyzed for pro-inflammatory and regulatory cytokine secretion profiles  with the BD Cytometric Bead Array System™ using a FACSCanto II® cytometer and appropriate FlexSets (BD Biosciences).This study was not designed to test for significant changes in PBMC measures or monocyte responses. The highly heterogeneous nature of the patient population in this study enabled investigators to simply monitor for minor trends that may occur following commencement of laquinimod therapy. Data from the two groups were calculated and visually assessed to demonstrate the outcome results after treatment with laquinimod 0.6 mg versus placebo.+CD4+ T cells, CD3+CD8+ T cells, CD4+CD25highFoxP3+ regulatory T cells, CD19+ B cells, CD56+ NK cells, CD14+ monocytes, and CD83+ mature dendritic cells. The measurements were performed in each participant and at each time point on freshly isolated PBMCs with the same protocol. This approach allowed for following individual immune parameters from the beginning to the end of the observation period. After 1, 3, 6, 12, and 24 months of laquinimod treatment, there were no significant quantitative changes in any of the analyzed immune cell populations including CD4+ T cells, CD8+ T cells, regulatory T cells, B cells, NK cells, NKT cells, monocytes, and mature dendritic cells. There were also no differences in any of the assessed PBMC populations between laquinimod treated individuals and controls receiving placebo at any time point  or recall antigen (tetanus toxin) in a me point . After 1On the basis of the results of our previous animal experiments indicating monocytes as a possible target of immune regulatory effects of laquinimod, we finally decided to perform a detailed analysis of monocyte phenotypes and cytokine secretion profile in vitro. To this end, monocytes freshly isolated from the peripheral blood of study participants were cultured under pro-inflammatory conditions with IFN-γ or LPS. After 72 h of culture cells were harvested for surface molecule and cytokine expression analysis. Over the course of the study, no influence of laquinimod was observed on the expression of MHC class II molecules neither under IFN-γ nor under LPS stimulation (data not shown). However, beginning form the 1st month of treatment, we observed a lower level of CD86 expression on monocytes stimulated with LPS in laquinimod treated patients  and 5. TIn addition to the observed in vitro changes on costimulatory molecules, laquinimod treatment also influenced the secretory profile of monocytes cultured in vitro. Throughout the study, the basal secretory profile of unstimulated monocytes in culture did not differ between placebo and laquinimod treated patients (data not shown). However, upon pro-inflammatory stimulation, monocytes obtained from laquinimod treated patients tended to secrete lower levels of proinflammatory chemokines: CCL2  and CCL5 . Interestingly, the down-regulation of CCL2 secretion was observed specifically after IFN-γ stimulation, whereas LPS stimulated monocytes demonstrated a lower production of CCL5 over the course of laquinimod treatment . We did In our study we prospectively analyzed a broad panel of cellular and humoral immune parameters ex vivo and in vitro in RRMS patients treated continuously for 2 years with laquinimod. The main finding of the analyses performed is a modulatory effect of laquinimod on the immune properties of monocytes stimulated in vitro with various pro-inflammatory factors including LPS and IFNγ, reflecting the immune potential of these cells in vivo.Already the earliest laquinimod studies in MS showed both protective and disease suppressive effects of this compound in different forms of EAE which is widely regarded as the best animal model of autoimmune CNS demyelination Recently, it was shown by our group that adoptive transfer of laquinimod treated monocytes in mice suffering from EAE ameliorated the clinical course of the disease in a mechanism most probably dependent on a monocyte shift towards a regulatory and neuroprotective immune profile associated with BDNF secretion Recently Jolivel et al. + DCs revealed no differences between the laquinimod and placebo treated groups over the 2 years of observation. Additionally, during the study we did not observe any effects of laquinimod treatment on the proliferative response of PBMCs either to mitogen or antigen-specific stimulation in vitro. These findings clearly show that longitudinal treatment with laquinimod does not exert any major, unspecific immunosuppressive influence on the crucial elements of patient's cellular immunity at any time point of the therapy, beginning from the 1st till the 24th month. Hence, we here provide a unique set of immune-safety data for the long-term treatment of MS patients with laquinimod. During the preparation of this manuscript, the results of a complementary study were published which solely focused on ex vivo analyses of immune cells from ALLEGRO study participants in the US In contrast to the in vitro findings, the ex vivo measurements encompassing all the main peripheral blood lymphocytic populations  as well as monocytes and mature CD83MS and RL planned and performed all the experimental procedures, LH designed the study and wrote the paper, OB analyzed the experimental data and performed the statistical analysis, RG designed the study and wrote the paper. The study was funded by Teva Pharmaceutical Industries Ltd, Israel.LH and OB are employees of Teva Pharmaceutical Industries Ltd, Israel.


As a result, this study seeks to prove the feasibility of combining a novel minimally invasive biopsy technique that expands the number of lesions amenable for biopsy with subsequent exvivo kinase activity analysis.Researchers are currently seeking relevant lung cancer biomarkers in order to make informed decisions regarding therapeutic selection for patients in so-called “precision medicine.” However, there are challenges to obtaining adequate lung cancer tissue for molecular analyses. Furthermore, current molecular testing of tumors at the genomic or transcriptomic level are very indirect measures of biological response to a drug, particularly for small molecule inhibitors that target kinases. Kinase activity profiling is therefore theorized to be more reflective of exvivo interrogation of samples in combination with tyrosine kinase inhibitors erlotinib, crizotinib, and lapatinib were performed by PamStation 12 microarray analysis.Eight patients with lung lesions of varying location and size were biopsied using the novel electromagnetic navigational bronchoscopy (ENB) technique. Basal kinase activity (kinomic) profiles and Kinomic profiling qualitatively identified patient specific kinase activity profiles as well as patient and drug specific changes in kinase activity profiles following exposure to inhibitor. Thus, the study has verified the feasibility of ENB as a method for obtaining tissue in adequate quantities for kinomic analysis and has demonstrated the possible use of this tissue acquisition and analysis technique as a method for future study of lung cancer biomarkers.We demonstrate the feasibility of using ENB-derived biopsies to perform kinase activity assessment in lung cancer patients. Novel therapies for lung cancer are being tested in both preclinical and clinical settings Kinomic profiling refers to the determination of global kinase activity in a specimen and is distinct from genomic and proteomic methods because it determines changes in biological activity, not just the presence of a gene, transcript, or protein As mentioned earlier, one of the limitations commonly faced in the clinic relates to the difficulty in obtaining adequate tissue for molecular analysis Electromagnetic navigational bronchoscopy (ENB) can potentially overcome some of these limitations by more reliably obtaining diagnostic material from almost any location in the lungs in sufficient quantities to allow for molecular testing This study was conducted in accordance with the Declaration of Helsinki and was approved by University of Alabama at Birmingham (UAB) Institutional Review Board (IRB# X110411014). Written informed consent was obtained from all patients.www.kinomecore.com) and stored at −80 C until the kinomic assay was performed. During the same ENB procedure, tumor specimen material was collected and provided to UAB Pathology when clinically indicated. In some cases, the patient already had a tissue diagnosis and underwent fiducial implantation for facilitation of stereotactic body radiation therapy treatment planning and delivery. These fiducials were placed after obtaining any biopsy material.ENB utilizes a specially formatted CT scan to create a virtual bronchoscopy. During the procedure the patient lies on an electromagnetic plate and chest wall sensors monitor patient movement with respiration. A standard video bronchoscope is used with a 3 mm working channel. An extended working channel and a locatable guide are passed through the bronchoscope’s working channel. The tip of the locatable guide is sensed in the electromagnetic field. Registration points are identified to match the virtual images to the patient's anatomy. The locatable guide is then navigated to the target lesion and biopsy specimens are taken under fluoroscopic visualization. For this study, the tissue specimens were collected in cryovials and immediately flash frozen in liquid nitrogen in the operating suite. These specimens were transferred directly to the UAB Kinome Core (exvivo drug testing if they had a) insufficient protein quantity, b) excessive temperature variation, c) insufficient basal kinase activity. Western blotting to measure relative levels of housekeeping proteins and total phospho-tyrosine was completed using 10% SDS-PAGE gels, and transferred to Immobilon PVDF membranes  with 20 ug of sample loaded per lane, and detection with 1∶4000 anti-phospho-tyrosine-HRP , 1∶5000 mouse anti-GAPDH and rabbit anti-actin  with horseradish-peroxidase conjugated goat anti-mouse, and donkey anti-rabbit antibodies .Frozen tissue for each tumor was processed at 4°C for kinomic profiling using eppendorf tube pestle grinding followed by lysis in M-PER lysis buffer with protease and phosphatase inhibitors per our standard protocol Ex vivo kinomics testing was performed using the small molecule kinase inhibitors, lapatinib, crizotinib, and erlotinib purchased from LC Laboratories . Ex vivo testing Primary analysis was performed using BioNavigator software v. 5.1 (PamGene).http://kinome.github.io/public.Kinase activity data were produced to correspond to the phosphorylatable-peptide specific reaction per ‘spot’ on the PamChip. Raw signal intensity data per each of the 144 spots was captured over multiple 50 ms exposures sequentially as lysates were pumped through the array, and then over multiple exposure times  after lysates were rinsed off. These values were converted to slopes of intensity by exposure time, and slopes were multiplied by 100 and log2 transformed for visualization and are labeled “log signal”. Heatmaps of log signal data were plotted in color corresponding with signal intensity  or with log change from control (drug response). Unsupervised hierarchical clustering of kinomic profiles for the samples was done using a Euclidean distance, complete linkage R-script within BioNavigator. Variance filtering was also performed on the log signal transformed data with deviation from peptide experimental mean per peptide in basal conditions. Dose-response heatmaps, are displayed as the log ratio change from a DMSO control. Selected peptides were plotted as raw kinetic values  over multiple 50 ms exposures to demonstrate specific treatment-altered phosphorylation curves. A publically available kinomic analysis tool containing the experimental data can be found at In this feasibility study, we consented and enrolled eight patients for kinomic testing of ENB-derived material , and allexvivo drug testing due to at least one of the exclusion criteria listed above. Pathologic evaluation of patient 2’s specimen indicated that the area biopsied was necrotic which would explain the relatively low level of kinase activity. Unfortunately, the specimens obtained from patients 3 and 5 were not evaluated by pathology due to prior diagnosis of non-small cell lung carcinoma (NSCLC). Of note, patient 3 had previously received definitive chemoradiation to the region but had evidence of progression by CT. Nevertheless, we cannot comment on the viability status of those specimens.Protein quantification of the samples indicated that the tissue specimens were of sufficient size to perform kinomic testing. Typical protein yield for an ENB biopsy sample obtained by micro-forceps was ∼378 µg/mL in approximately 150 µL of total volume. Since kinomic testing requires between 1–5 µg of total protein depending on array type (the serine-threonine kinome [STK] chip requiring 1 µg and the tyrosine kinome [PTK] chip requiring 5 µg), the samples obtained were more than adequate. Variation in loading control protein expression did not always linearly correlate with measured total protein concentrations. Although there was adequate protein, patients 2, 3, and 5 were not suitable for http://kinome.github.io/public). All 8 patients were analyzed as this was a feasibility study, though only 2 of the 5 patients that had simultaneous pathology review had viable cancer cells. There was a large variation in mean log signal intensity among the samples consistent with the variety of tissues obtained . We measured a maximum peptide log signal variance of 25.86, with mean peptide variance of 9.777. The standard deviation from the experimental all-peptide mean  was 2.54 with the highest signal sample having a mean log ratio change from the experimental mean of +4.13(PAT1) and the lowest had a −2.02 (PAT7) log ratio change. Technical variance  was measured across all peptides  at 2.7% CV. This was inline with previous experiments where we had seen median technical and biological variance in controlled cell lysates measured at 4.2% and 4.6% CV (data not shown).The basal kinomic profiles of all 8 patients are shown as unsupervised hierarchically clustered heatmaps of log signal intensity for each phosphosubstrate  during bronchoscopy procedures, including ENB. This “optical biopsy” can improve diagnostic yields since neoplastic surface mucosa can be more readily distinguished from non-neoplastic mucosa The basal heatmap  intimateexvivo small molecule inhibitor testing  versus normal tissue to allow for confident identification of relevant tumor markers. Additionally, there are inadequate numbers of patients to build meaningful models predictive of treatment response. Since some patients did not have a specimen sent for pathological evaluation, we also cannot determine whether the samples with low global kinomic activity was due to the presence of non-viable tissue. As with standard biopsy collection methods, a limitation in molecular analysis is that tumors are highly heterogeneous, even within a single solid tumor, therefore making extrapolations from single biopsies to the entire tumor’s biology is difficult. Comparative multi-site intra-tumor sampling analyses and comparison between ENB-derived tissues and standard biopsy derived tissues in the same patient are generally not feasible from a clinical standpoint. Although this study provides conceptual proof of the feasibility of prospective kinomic analysis of ENB derived tissue, further research will be necessary to investigate the application of this technique to the “precision medicine” landscape of lung cancer.This study demonstrates the feasibility of kinase activity microarray testing of tissue derived by electromagnetic navigational bronchoscopy.S1 FigAnti-phosphotyrosine Western blot was performed for total protein lysates from patient #1 and #6 (PAT1 and PAT6) using 1∶3000 HRP-conjugated anti-phosphotyrosine antibody with anti-actin as a loading control.(TIF)Click here for additional data file.S2 FigA–C) above tables of their respective Kinexus identified and ranked upstream ‘Kinases’ and Uniprot ID’s. The percentage of times a kinase was present in a top-ten list upstream of a phosphorylatable residue in the substrate is listed as ’%hits’. These upstream kinases (red circles on network maps) identified were uploaded by Uniprot ID to GeneGo MetaCore for network modeling .Top upstream kinases and network maps for each of the 3 differentially phosphorylated substrates shown in (TIF)Click here for additional data file.