Gravin−/− mice where an overabundance of Oct3/4 positive germ line stem cells displays randomized orientation of mitotic spindles. Thus, we propose that Gravin-mediated recruitment of Aurora A and Plk1 to the mother (oldest) spindle pole contributes to the fidelity of symmetric cell division.Correct orientation of the mitotic spindle in stem cells underlies organogenesis. Spindle abnormalities correlate with cancer progression in germ line-derived tumors. We discover a macromolecular complex between the scaffolding protein Gravin/AKAP12 and the mitotic kinases, Aurora A and Plk1, that is down regulated in human seminoma. Depletion of Gravin correlates with an increased mitotic index and disorganization of seminiferous tubules. Biochemical, super-resolution imaging, and enzymology approaches establish that this Gravin scaffold accumulates at the mother spindle pole during metaphase. Manipulating elements of the Gravin-Aurora A-Plk1 axis prompts mitotic delay and prevents appropriate assembly of astral microtubules to promote spindle misorientation. These pathological responses are conserved in seminiferous tubules from DOI:http://dx.doi.org/10.7554/eLife.09384.001 The genetic material inside our cells is contained within structures called chromosomes. When a cell divides, these chromosomes are copied and then must be correctly divided between the two daughter cells so that each cell has a complete set of genetic material. The correct separation of the chromosomes depends on a structure called the mitotic spindle whose location in the cell also determines where the point of division will be.Two structures called centrioles are associated with the mitotic spindle and help to organize and direct cell division. The cell carefully controls how these structures are inherited by the daughter cells. For example, when a stem cell divides to produce one stem cell and one cell of a different type, the older centriole can be inherited by the new stem cell. Incorrect placement of the spindle can disrupt this process and is linked to the progression of cancers that affect reproductive organs, such as testicular seminomas.Here, Hehnly, Canton et al. used biochemical and microscopy techniques to study how the spindle is positioned in cells from patients with testicular seminoma tumors. The experiments reveal that a protein called Gravin is found in lower amounts in the tumor cells than in normal cells. In mice that lack Gravin, the cells in a region of the testicle called the seminiferous tubule divide more rapidly, which is a hallmark of cancer. Gravin accumulates at the end of the spindle where the older centriole is present. This protein acts as a scaffold that holds two enzymes called kinases that regulate cell division in place at the end of the spindle. In the stem cells of the testicle, these kinases also appear to help to correctly position the spindle by organizing the proteins that anchor this end of the spindle to the membrane.Hehnly, Canton et al.'s findings suggest that Gravin helps to guard against errors occurring during cell division by recruiting two particular kinase enzymes to the mitotic spindle. A future challenge will be to identify the proteins that these kinases affect while anchored to the spindle.DOI:http://dx.doi.org/10.7554/eLife.09384.002 Mitotic cell division is a process whereby genetic material is duplicated, separated, and packaged to yield two daughter cells . This prThe mitotic spindle is constrained by two spindle poles that nucleate microtubules. The mother spindle pole contains the oldest centriole and remains anchored near the stem-cell niche, while the daughter spindle pole migrates to the opposite side of the cell to complete spindle formation . RecentlSpindle orientation defects that promote an imbalance between symmetric and asymmetric cell divisions have been implicated in the progression of germ line-derived cancers such as teratomas, seminomas, and ovarian carcinomas . These cMutation or amplification of Gravin has been linked to melanoma, prostate, and ovarian cancers, yet nothing is known about the role of this kinase-anchoring protein in solid tumors . TesticuImmunofluorescent screening of seminoma tumor sections confirmed a reduction in Gravin . In normFurther evidence that Gravin loss alters mitotic progression was obtained from knockout mice . SeminifOur initial findings postulate that Gravin loss contributes to the mitotic abnormalities observed in seminoma. One plausible explanation is that loss of Gravin uncouples the location of protein kinases that drive the cell cycle. Phosphorylation of Gravin on Threonine 766 promotes recruitment of Plk1, a kinase that prompts mitotic progression and appropriate spindle formation . A key aTo test this concept, four complementary approaches were used. First, immunoblot analysis detected Aurora A and Plk1 in Gravin immune complexes isolated from mitotic cell lysates , lane 6.Since p766-Gravin, Aurora A, and Plk1 decorate mitotic spindle poles, we reasoned that the anchoring protein may actively constrain both enzymes at this location. To test this hypothesis, the subcellular distribution of both kinases was evaluated in MEFs from wild-type and Gravin knockout mice . MetaphaAurora A and Plk1 are required for spindle pole maturation . One aspMitotic spindle poles are classified as either the mother spindle pole that contains material that formed the original centriole or the more recently assembled daughter spindle pole . Upon clGSDIM was also employed to test whether the asymmetric distribution of Plk1 to the mother spindle pole required Gravin . SurprisWe reasoned that one consequence of the aforementioned result could be that differential localization of the Gravin scaffold to the mother spindle pole favors more robust assembly of astral microtubules. In order to test this hypothesis, we evaluated astral microtubule abundance, length, and ultrastructure at super-resolution using SIM . PericenWe propose that Aurora A and Plk1 are assembled into a kinase cascade at the mother spindle pole through their simultaneous association with p766-Gravin. Cellular and molecular validation of this model was conducted in three phases. First, Aurora A/Plk1 PLA pairs were localized throughout the cell with a subpopulation accumulating at one mitotic spindle pole . Importa3/IP) blocked by the inhibitor drug VX-680  was measured in Gravin immune complexes isolated from mitotic lysates. Aurora A kinase activity was defined as the number of counts  that directly interacts with Aurora A  created To define the Gravin-Plk1 interface during mitosis, we monitored cells depleted of Gravin and rescued with kinase-binding mutants of the anchoring protein. Metaphase was delayed in Gravin shRNA depleted cells  compared to control  residing on the inner face of the basement membrane . ApproprDuring self-renewal, a mitotic cell chooses to orient its spindle either parallel or perpendicular to the basement membrane . Self-reOn the basis of our cell-based and biochemical studies in Two possible outcomes can arise from the loss of Gravin expression in stem cells. Existing stem cell populations that undergo misoriented division may expand exponentially. Alternatively, germ line stem cells may prematurely differentiate thereby diminishing the total stem cell population. In order to delineate between these two possibilities, we counted the number Oct3/4-positive cells in seminiferous tubule sections . A 3.82-Aurora A and Plk1 are two kinases generally considered to act together to promote spindle pole maturation . We haveSpindle maturation occurs during the transition from prophase to metaphase when dynein-mediated transport of PCM along the microtubules culminates in the formation of a mitotic spindle . SurprisOne new concept that emerges from our studies is that Gravin-mediated clustering of Aurora A and Plk1 at the mother spindle pole provides a means to more precisely regulate symmetric cell division. We offer four lines of inquiry to support this new mechanism. First, data presented in Another theme emerging from the data in Akap12 locus) mice were generated as described in , rabbit anti-pericentrin , goat aribed in  and obtaTransIT-LTI reagent (Mirus) for Hek293 cells, Hela monster (Mirus) for Hela cells, or by nucleofection using Ingenio (mirus) for RPE cells.Hela cells, U2OS, and MEFs  were maintained in D (Dulbecco's)-minimal essential medium (MEM) and retinal pigment epithelial cells (RPE) were maintained in DMEM:F12. All media was supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin/streptomycin, and 1% Glut-MAX (Invitrogen). Infections for generation of stable knockdowns were performed with shRNA lentiviral particles (Santa Cruz Biotech) or retroviral particles . Transient gene expression was performed by transfection using MEFs were isolated following the protocol provided by . BrieflyAll human specimens were purchased from BioChain Institute, Inc. Reproductive age male mice (∼7 weeks of age) were sacrificed, testes were removed, fixed in formalin for >24 hr at 4°, and embedded in paraffin. Samples were sectioned at 5 μm, mounted onto slides, and subjected to H&E or conventional antigen retrieval through deparaffination followed by immunostaining. Sections were deparaffinized, rehydrated, and incubated with antibodies as labeled.Images for spindle tilt, tissue sections, and general spindle morphology were acquired using primarily a Yokogawa CSU10 spinning disk mounted on a DM16000B inverted microscope  with an Andor ILE laser launch with 50 mW Coherent OBIS lasers  unless otherwise noted in the manuscript. Two separate cameras were used depending on whether it was live-cell acquisition (Hamamatsu ImagEM EM-CCD Camera C9100-13) or fixed samples . Z-stacks were shown as 2D maximum projections or processed for 3-dimensional rendering (Metamorph). Fluorescence range intensity was adjusted identically for each series of panels. Intensity profiles and fluorescence intensity quantification were obtained from sum projections of Z stacks using either Metamorph or ImageJ/Fiji software. Fluorescence intensity quantification of spindle poles was carried out as previously described . In shorCoverslips that were fixed and stained with primary antibodies towards Plk1, Aurora A, Cenexin, Centrobin, p-Gravin (T766A), and Gravin for 1 hr and followed with secondary antibodies (Alexa Fluor 647 or Alexa Fluor 568). Coverslips were mounted with MEA-GLOX imaging buffer  on glass depression slides  and sealed with Twinsil . Ground state depletion (GSD) super-resolution images of mitotic spindle poles were generated using a Leica SR GSD 3D system. The system is built around a Leica DMI6000 B TIRF microscope and is equipped with a Leica oil-immersion HC PL APO 160×/1.43 NA super-resolution objective, four laser lines , and an Andor iXon3 EM-CCD. Images were collected in epifluorescent mode at a frame rate of 100 Hz for 50,000–100,000 frames using Leica Application Suite (LAS AF) software. Intensity calculations and 3-dimensional heatmaps were done in ImageJ/Fiji.Super-resolution 3D-SIM images were acquired on a DeltaVision OMX V4  equipped with a 60×/1.42 NA PlanApo oil immersion lens (Olympus), 405-, 488-, 568-, and 642-nm solid-state lasers and sCMOS cameras (pco.edge). Image stacks of 5–6 µm with 0.125-µm thick z-sections and 15 images per optical slice (3 angles and 5 phases) which were acquired using immersion oil with a refractive index 1.518. Images were reconstructed using Wiener filter settings of 0.003 and optical transfer functions measured specifically for each channel with SoftWoRx 6.1.3  to obtain super-resolution images with a twofold increase in resolution both axially and laterally. Images from different color channels were registered using parameters generated from a gold grid registration slide  and SoftWoRx 6.1.3 .Staining procedures for cultured cells were performed as previously described .Procedures were performed as described in .Cells were cultured on 35-mm dishes containing a central 14 mm 1.5 glass coverslip (MatTek). The cells were imaged in DMEM without phenol plus 20 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) and 10% FBS at 37°C. Spinning disk confocal microscopy was performed on the above system attached to a Hamamatsu ImagEM, EM-CCD Camera C9100-13. For GFP imaging, frames were acquired every 2 to 5 min with an exposure time of 100 ms.Determination of spindle orientation relative to the horizontal plane or basement membrane in seminiferous tubules was performed as previously described . BrieflyProcedures were done as described in t-test or unpaired with Welch's correction or Mann–Whitney U-test, for two-group comparisons using Prism's Graph Pad. p-values less than 0.05 were considered statistically significant.Statistics were performed using paired Student's  review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  and three reviewers, one of whom is a member of our Board of Reviewing Editors.Thank you for submitting your work entitled “A mitotic kinase scaffold depleted in testicular seminomas impacts spindle orientation in germline stem cells” for peer review at The reviewers have discussed the reviews with one another and the Reviewing editor has drafted this decision to help you prepare a revised submission.Hehnly et al. report that Gravin is required for proper spindle orientation in germ line stem cells during spermatogenesis and is depleted in seminomas. Using a combination of techniques in a variety of cell types, the authors have demonstrated that Gravin interacts with Aurora A and Plk1, and recruits these kinases at the mother spindle pole for proper assembly of astral microtubules. The data presented are interesting and the manuscript is well organized. However, the mechanisms by which Gravin scaffold accumulation at the mother spindle pole ensures proper spindle orientation in germline stem cells remain unclear.−/− cells (1) The authors propose that Gravin ensures symmetric, but not asymmetric, cell division (Introduction). However, it is unclear whether Gravin is critical for successful symmetric or asymmetric stem cell division. First, Oct3/4 positive stem cells are increased in Gravin/− cells . As symm/− cells .2) The authors demonstrate that there are two populations of dividing germ line stem cells in 3) The authors demonstrate that p766-Gravin, Aurora A and Plk1 accumulate at the mother spindle pole in MEFs . However−/− MEF grew more slowly than wt MEF. In contrast, analysts of seminiferous tubule sections demonstrated cell with increased mitotic index. The authors need to clarify these observations – do both cell types grow slowly in the absence of gravin and the increased mitotic index simply a reflection of a mitotic delay. Or does Gravin have opposite phenotypes in the cells?4) The authors conclude that Gravin is important for mitotic progression. Gravin−/− MEF show decreased Plk1 and AuroraA? If so, does the loss of total protein explain the decreased spindle pole localization? Does loss of protein expression account for the role of Gravin to mediate interactions between Plk1 and AuroraA.5) The immunoblot analysis of tumors  shows lo6) The rescue experiments using the T766A mutant of Gravin should be extended to demonstrate that it is unable to recruit Plk1 to the mother spindle pole to regulate astral microtubules.7) The authors state data as xxx {plus minus} yyy in several places in the text. The numbers should be defined. Is xxx a mean or a median or something else? Is yyy the SD, SEM or something else? Also, do the statistics justify the precision of the numbers provided – for example, 64.57 {plus minus} 4.09 (in the first paragraph of subsection “Gravin anchoring of Aurora A and Plk1 is required for mitotic progression”). 1) The authors propose that Gravin ensures symmetric, but not asymmetric, cell division (Introduction). However, it is unclear whether Gravin is critical for successful symmetric or asymmetric stem cell division. First, Oct3/4 positive stem cells are increased in Gravin−/−cells . As symmetric stem cell division produces two daughter stem cells (subsection “Gravin contributes to spindle orientation of germline stem cells during spermatogenesis”), defects in parallel symmetric cell division would lead to detachment of one daughter cell from the basal membrane and an expected decrease in stem cell population.Gravin−/− mice. In wildtype cells quantitative analysis of tissue sections stained for tubulin reveals two distinct populations of spindle organization. In contrast, we detect a randomized spindle orientation in Gravin null tissue  in Gravin shRNA treated cells as compared to controls  indicating that signaling events downstream of Plk1 modulate the correct orientation of mitotic spindles. However a vital new piece of this puzzle, solely uncovered from our work, is that Gravin functions as the anchor for these enzymes. Interpretation of the new data in 2) The authors demonstrate that there are two populations of dividing germ line stem cells in. It is critical to analyze which populations contain phosphorylated Gravin, althoughshows that p766-Gravin signals are detected in symmetrically dividing self-renewing cells. In addition, it is important to analyze if p766-Gravin accumulates at the mother spindle pole in the germ line stem cells similar to MEFs.We have responded to the reviewers' suggestions in three ways: i) p766-Gravin is present in all germline stem cells and wildtype MEFs during mitosis, irrespective of which direction either cell-type divides. To emphasize this point we have included an additional representative image of p766-Gravin in ii) Perhaps not surprisingly, the asymmetric distribution of p766-Gravin between the mother and daughter spindle poles is most evident upon analysis by super resolution microscopy. In keeping with this statement we now include a comparison between data collected by conventional wide field microscopy and structured illumination microscopy in −/− MEFs. We contend these cell lines represent a valid model system that recapitulates elements of the in vivo environment. Particularly since we observe the same randomization of mitotic spindle angles upon loss or depletion of Gravin in all systems (iii) Finally, we wish to point out that super resolution microscopy is technically challenging and often prohibitive when attempted on paraffin embedded tissue samples such as the sections used in  systems . This ne3) The authors demonstrate that p766-Gravin, Aurora A and Plk1 accumulate at the mother spindle pole in MEFs . However, the exact contribution of spindle pole asymmetry to astral microtubule formation is still unclear. The authors need to compare the astral microtubules derived from mother and daughter spindle poles, respectively, as shown in−/− cells  The authors conclude that Gravin is important for mitotic progression. Gravin−/−MEF grew more slowly than wt MEF. In contrast, analysts of seminiferous tubule sections demonstrated cell with increased mitotic index. The authors need to clarify these observations – do both cell types grow slowly in the absence of gravin and the increased mitotic index simply a reflection of a mitotic delay. Or does Gravin have opposite phenotypes in the cells?−/− seminiferous tubules  The immunoblot analysis of tumors  shows loss of Gravin, but also loss of Plk1 and AuroraA. Are these related events? Do Gravin−/−MEF show decreased Plk1 and AuroraA? If so, does the loss of total protein explain the decreased spindle pole localization? Does loss of protein expression account for the role of Gravin to mediate interactions between Plk1 and AuroraA.Immunoblot data of clinical samples from seminoma patients presented in −/− MEFs the data were inconclusive. The Plk1 immunoblot signal was reduced once, constant in one instance, and slightly increased in the final attempts. Thus, we were forced to conclude that although the Aurora A and Plk1 antibodies are reliable for immunofluorescent detection of the human and murine kinases, they are less cross reactive with murine orthologs when used for immunoblot detection. As a backup approach we monitored the levels of the human Aurora A and Plk1 upon shRNA deletion of Gravin in HEK293 cells.Despite eleven independent attempts, immunobot detection of the murine Plk1 and Aurora A orthologs in MEFs was sporadic. A majority of the time we were unable to detect either kinase by immunoblot. Yet on the few occasions when we were able to detect any Plk1 signal in mitotic GravinNew data shows that gene silencing of Gravin had little or no effect on the levels of Aurora A or Plk1 during mitosis (6) The rescue experiments using the T766A mutant of Gravin should be extended to demonstrate that it is unable to recruit Plk1 to the mother spindle pole to regulate astral microtubules.−/− MEFs is unable to rescue the number of astral microtubules protruding form the mitotic spindle. Rather, rescue with this Plk1-anchoring defective mutant may exacerbate the astral microtubule phenotype. These data are included in We have previously shown that the Gravin T766A mutant is unable to bind Plk1 in a variety of cell types. This information has been added to the text and the appropriate papers citied. In addition new data presented in 7) The authors state data as xxx {plus minus} yyy in several places in the text. The numbers should be defined. Is xxx a mean or a median or something else? Is yyy the SD, SEM or something else? Also, do the statistics justify the precision of the numbers provided – for example, 64.57 {plus minus} 4.09 (in the first paragraph of subsection “Gravin anchoring of Aurora A and Plk1 is required for mitotic progression”).We have clarified these points. In all cases where a bar graph is presented or where a scatter plot is presented XXX represents the mean and the +/- demonstrates the range in standard error of the mean. We have checked all the exact means added to the text and made sure they are consistent with the data presented. In the cases where we present a Box and Whiskers plot, the box extends from the 25th to 75th percentiles using a standard method of computation via Prism software. The line in the middle of the box is plotted as the median.


The Trial of Early Aggressive Therapy in Juvenile Idiopathic Arthritis  was accompanied by a once-in-a-generation sample collection for translational research. In this paper, we report the results of whole blood gene expression analyses and genomic data-mining designed to cast light on the immunopathogenesis of polyarticular juvenile idiopathic arthritis (JIA).TREAT samples and samples from an independent cohort were analyzed on Affymetrix microarrays and compared to healthy controls. Data from the independent cohort were used to validate the TREAT data. Pathways analysis was used to characterize gene expression profiles. Furthermore, we correlated differential gene expression with new information about functional regulatory elements within the genome to develop models of aberrant gene expression in JIA.There was a strong concordance in gene expression between TREAT samples and the independent cohort. In addition, rheumatoid factor (RF)-positive and RF-negative patients showed only small differences on whole blood expression profiles. Analysis of the combined samples showed 158 genes represented by 176 probes that showed differential expression between TREAT subjects at baseline and healthy controls. None of the differentially expressed genes were encoded within linkage disequilibrium blocks containing single nucleotide polymorphisms known to be associated with risk for JIA. Functional analysis of these genes showed functional associations with multiple processes associated with innate and adaptive immunity, and appeared to reflect overall suppression of STAT1–3/interferon response factor-mediated pathways.Despite their limitations, whole blood expression profiles clearly distinguish children with polyarticular JIA from healthy controls. Whole blood expression profiles identify several immunologic pathways of biologic relevance that will need to be pursued in homogeneous cell populations in order to clarify mechanisms of pathogenesis.#NCT00443430, originally registered 2 March 2007 and last updated 30 May 2013.ClinicalTrials.gov registry  PatientControls consisted of 8 healthy girls and 11 healthy boys between the ages of 7 and 13 years recruited from the OU Children’s Physicians General Pediatrics clinic. The protocol for obtaining these specimens was approved by the University of Oklahoma IRB (#13205). Anesthesia for the phlebotomy was provided using topical lidocaine/prilocaine solution. These samples are hereafter referred to as healthy children (HC).In addition to the above, whole blood PAXgene specimens were obtained from an independent cohort of children with newly diagnosed, polyarticular-onset JIA recruited from the University of Oklahoma Health Sciences Center pediatric rheumatology clinic. These children ranged in age from 3 years to 15 years and consisted of 4 boys and 6 girls. All patients in this cohort were rheumatoid factor (RF)-negative. These samples are hereafter referred to as the Oklahoma cohort (OK). Table All research procedures were carried out strictly following the IRB-approved protocols.RNA was purified from whole blood PAXgene specimens using a PAXgene Blood RNA kit  as recommended by the manufacturer, including a DNAse (Qiagen) step to remove genomic DNA. Globin transcripts, which reduce labeling efficiency of whole blood cell RNA and decrease signal-to-noise ratios on microarrays , were recRNA was produced from reverse transcribed cDNA using the Illumina® TotalPrep RNA Amplification Kit , hybridized to Illumina WG-6 v3 or Illumina HT-12 v4 human whole genome microarrays, and stained according to the manufacturer’s directions. Array hybridizations were undertaken in three separate batches. The first batch consisted of the 19 healthy controls and 26 baseline samples hybridized on Illumina WG-6 v3 arrays. The second batch consisted of the remaining patient samples hybridized to Illumina HT-12 v4 arrays. The independent cohort of OK samples were hybridized on Illumina WG-6 v3 slides. Gene microarray data have been made available to the scientific public (GEO Accession Number GSE55319).www.r-project.org). To facilitate statistical analyses relative to healthy controls, it was necessary to combine data from different array batches. Due to the difference in the arrays it was necessary to create combined datasets using only those probes that were present on both array formats. Illumina probe IDs were used to identify 39,426 common probes. Datasets were variance stabilized and normalized using robust spline normalization via the lumi package [p value provided by the Illumina quality control metrics to eliminate probes not responding at higher than background levels.All statistical analyses were carried out in R  that indicates the probability of the query genes (defined as target molecules in IPA) in a network being randomly associated within a connecting network. It is important to note that the network score does not infer network quality; it simply indicates the fitness between a reference network and the network of query genes.We used the Ingenuity Pathway Analysis (IPA) software  for network analysis, with differentially expressed (DE) genes as input following the default setting of a maximum 35 genes per network. IPA network generation transforms the query genes into a set of relevant networks based on its extensive, curated Ingenuity Pathway Knowledge Base database , 13. IPAhttp://www.broadinstitute.org/mpg/snap) using the 1000 genome project pilot1 and HapMap3 with a cutoff of r2 < 0.9 and a distance limit of 500 bases.The recent completion of a genome-wide fine mapping study for JIA  extends From the TREAT study we analyzed 44 baseline samples from 28 RF-negative patients and 16 RF-positive patients, plus 19 control samples from healthy children. In addition, we analyzed independent samples from 10 RF-negative patients (the OK cohort). Oklahoma and TREAT data were first normalized without using the COMBAT algorithm that we previously applied to the TREAT data to filter out batch effects [We first created a scatter plot correlating the OK and TREAT expression values for all probes Fig. . As showp value representing the confidence that a given transcript is expressed above the background defined by negative control probes. The cut-off we used for trimming probes from the arrays prior to differential expression analysis was to only select probes with p ≤ 0.01. The histograms in Fig. p value ≤0.01. Using this approach, we identified multiple poor quality probes on the both array sets.We then asked whether those probes that are low in one set but high in the other may reflect poor quality in the arrays, especially for low-expression probes. To investigate this possibility, we used Illumina array GenomeStudio software, which calculates and reports a detection r2 (0.627 for our two datasets).Upon splitting the probes into groups based on their quality, we discerned that there were technical issues creating the two unmatched tails. In the comparison, we required that all 10 of the Oklahoma arrays be good quality Fig. . Using tWe also note that in our biomarker paper  we were n = 16) and RF-negative (n = 28) samples. We found only 7 genes represented on 8 probes that showed differential expression between these groups; these genes are shown in Table We first compared RF-positive .The search for overlaps between the DE genes identified in the comparison between healthy controls and baseline TREAT subjects and LD blocks containing JIA-associated SNPs yielded no DE genes within any of the observed LD blocks. We next determined the closest LD blocks to the DE genes, and identified 75 LD blocks containing JIA-associated SNPs situated near 38 DE genes, with distances ranging between 345 and 390 kilobases. Next, we interrogated the upstream regulators of the DE genes as identified on Ingenuity analysis, i.e., IRF1, IRF3, IRF5, STAT1, STAT2, and STAT3. We queried the LD blocks containing the JIA-associated SNPs using the bedtools program. We downloaded ENCODE transcription factor binding site (TFBS) data from UCSC Genome Browser ENCODE data at In this paper, we analyzed the TREAT whole blood gene microarray data in an attempt to gather insights into disease mechanisms in polyarticular JIA. We found that the whole blood expression profiles reflect complex interactions between innate and adaptive immunity, a finding that is consistent with our previous reports that both peripheral blood mononuclear cell (PBMC)  and neutThese findings are also significant for what they do not tell us. Among the 158 genes that showed differential expression between baseline TREAT samples and those of healthy controls, none was located within LD blocks where there is known genetic risk for JIA , 15, norOur findings here suggest potentially useful targets for therapy in JIA. For example, the broad suppression of type 1 and type 2 interferon responses in JIA may reflect overall suppression of TLR9-mediated processes. TLR9 is an intracellular pattern recognition receptor that detects highly methylated DNA, which is common in bacterial and viral genomes  . In receThere are obviously limitations to these data and their interpretation. The first is the inherent “noisiness” of whole blood expression profiles. Whole blood expression profiles represent an amalgam of peripheral blood cells, including abundant leukocyte subtypes such as neutrophils and platelets, and less abundant cells such as monocytes, natural killer (NK) cells, and even circulating CD34+ cells . Furthertrans-acting enhancers [Whole genome expression profiling of untreated children with polyarticular JIA reveals complex transcriptional differences when compared with healthy controls. Activation of leukocyte chemotaxis/extravasation pathways and neutrophil activation by CSF3 are reflected in whole blood transcription analyses. At the same time, suppression of STAT1–3/IRF pathways, as we have previously reported in JIA neutrophils , is revenhancers ) than caDE, differentially expressed; ET, etanercept; FDR, false discovery rate, HC, healthy children; IL, interleukin; IPA, Ingenuity Pathway Analysis; IRF, interferon response factor; JIA, juvenile idiopathic arthritis; LD, linkage disequilibrium; MTX, methotrexate; NIH, National Institute of Health; OK, independent Oklahoma cohort; PBMC, peripheral blood mononuclear cell; RF, rheumatoid factor; SNP, single nucleotide polymorphism; TFBS, transcription factor binding site; TGF, transforming growth factor; TLR, Toll-like receptor; TREAT, Trial of Early Aggressive Therapy in Juvenile Idiopathic Arthritis


Multielectrode arrays (MEA) are broadly used for in vitro cell culture observation. Complete realization of this platform’s potential to the study of disease and development depends on the capability to make 1) reliable and repeatable observations, 2) neuronal network behavior characterizations, and 3) meaningful comparisons between cultures. Using an extensive, previously published, set of recordings [We utilize 878 recordings from embryonic rat cortex cell cultures collected from 60-electrode, grid-type 200 μm) MEA’s. We modeled each recording as a 60-node directed weighted graph with weights describing electrode connectivity and nodal clustering coefficients  as featu00 μm MEABias from defective electrodes can be avoided wholly by proper documentation of experimental conditions. This highlights the need for best practices when recording MEA signals. Our analysis highlights possibilities for post-experimental review to identify unknown issues and retroactively handle them. Thereupon, we make a case for transparency in data reporting and propose best practices for experimental and analysis phases.


Aspergillus nidulans and probably in other fungi of the pezizomycotina subphylum. A second component of this pathway has been identified and characterized. SltB is a 1272 amino acid protein with at least two putative functional domains, a pseudo-kinase and a serine-endoprotease, involved in signaling to the transcription factor SltA. Absence of SltB activity results in nearly identical phenotypes to those observed for a null sltA mutant. Hypersensitivity to a variety of monovalent and divalent cations, and to medium alkalinization are among the phenotypes exhibited by a null sltB mutant. Calcium homeostasis is an exception and this cation improves growth of sltΔ mutants. Moreover, loss of kinase HalA in conjunction with loss-of-function sltA or sltB mutations leads to pronounced calcium auxotrophy. sltA sltB double null mutants display a cation stress sensitive phenotype indistinguishable from that of single slt mutants showing the close functional relationship between these two proteins. This functional relationship is reinforced by the fact that numerous mutations in both slt loci can be isolated as suppressors of poor colonial growth resulting from certain null vps (vacuolar protein sorting) mutations. In addition to allowing identification of sltB, our sltB missense mutations enabled prediction of functional regions in the SltB protein. Although the relationship between the Slt and Vps pathways remains enigmatic, absence of SltB, like that of SltA, leads to vacuolar hypertrophy. Importantly, the phenotypes of selected sltA and sltB mutations demonstrate that suppression of null vps mutations is not dependent on the inability to tolerate cation stress. Thus a specific role for both SltA and SltB in the VPS pathway seems likely. Finally, it is noteworthy that SltA and SltB have a similar, limited phylogenetic distribution, being restricted to the pezizomycotina subphylum. The relevance of the Slt regulatory pathway to cell structure, intracellular trafficking and cation homeostasis and its restricted phylogenetic distribution makes this pathway of general interest for future investigation and as a source of targets for antifungal drugs.The transcriptional response to alkali metal cation stress is mediated by the zinc finger transcription factor SltA in  Fungi have developed genetic-based strategies to tolerate a wide range of environmental stresses such as variations in pH, temperature, nutrient availability, reactive oxygen species, osmolarity and ionic strength. The ambient pH response in fungi has been thoroughly studied in many species because of its general importance for viability, adaptation and pathogenicity. For example, secreted proteins  and metabolites  are useful only if produced at pHs reasonably near their maximal activity . NutrienAspergillus nidulans, tolerance of alkaline ambient pH requires the activities of, at least, three zinc-finger transcription factors: PacC, CrzA and SltA (Saccharomyces cerevisiae) mediates changes in gene expression in response to neutral or alkaline pH  is a phospho-protein. Different phosphorylation states have been identified in response to elevated calcium levels or alkaline pH  in A. nidulans, which encodes a transcription factor with three zinc fingers of the C2H2 class   . sltA dekalinity . In contkalinity . Ace1, ajecorina , is the jecorina .vcxA, encoding a vacuolar calcium exchanger and a positive effect on the Na+ pump ATPase-encoding enaA have been described  alleles  and consequently Asn247His. The pabaB22 mutation of strain MAD3682 . Strains were cultured in standard Aspergillus minimal (AMM) and complete (ACM) media , or for mutant sltB strains based on reversal of sensitivity to elevated concentrations of potassium (0.6 M KCl) in the media. Other selection procedures are specifically described in the text . Construction of the genomic DNA library in the pRG3-AMA1 plasmid and its use in the identification of genes by complementation is described in  MAD3682  was compM) media  at 37 °CsltA, sltB and pskA.Genomic DNA and total RNA extraction for northern and Southern blotting were carried out following standard protocols as described in 2.2sltB, sltA and pskA deletion cassettes were constructed as gene-replacement cassettes containing the riboB or pyrG gene of Aspergillus fumigatus as the selection marker by fusion PCR (vpsΔ strains were obtained from transformation plates following the procedure described in sltA and sltB mutations were identified by sequencing.The sion PCR . Primers2.32 concentration where indicated. Mycelia were incubated for one hour and samples were collected at time 0 (absence of added NaCl or CaCl2), 15, 30 and 60 min (presence of NaCl or CaCl2 where indicated). Total RNA extraction and northern protocols followed (sltA and sltB were detected using specific genomic probes amplified by PCR (oligonucleotides listed in sltA and a 1452 bp probe (covering 35% of the ORF) was used for sltB. Transcript levels of the housekeeping glyceraldehyde-3-phosphate dehydrogenase gene (gpdA) was used as internal control for normalization. A PhosphorImager imaging plate, BAS-IP-MS 2040, was used to detect radioactive mRNA bound probes with development using a FLA-5100 Reader (FujiFilm). Quantification of band intensities was performed using Multi-Gauge V3.0 software (Fujifilm).Wild-type and mutant strains were cultivated in supplemented liquid AMM for 16 h at 37 °C, after which the culture medium was made 1 M with respect to NaCl concentration or 10 mM with respect to CaClfollowed . Transcr2.4A. nidulans strains were inoculated in watch minimal medium -tartrate and 5 mM glucose and grown for 16–18 h at 25 °C. Staining of vacuoles with CMAC blue  and external and internal membranes with FM4-64  was as described in Conidiospores of ium WMM,  with 17.2.5http://mobyle.pasteur.fr/). Phylogenetic trees were generated using Mega 5.2 software (http://merops.sanger.ac.uk/) and the clustalw2 application in EBI  were used for sequence similarity searching and analysis of the SltB protease domain.Multiple sequence alignments were made using the ClustalX program . Alignmesoftware , followi33.1vps31ΔbroA strain. This mutation, now designated sltB2 arose at some point in the course of construction, growth or haploidization of a diploid heterozygous for vps31ΔbroA. BroA is the homologue of S. cerevisiae Bro1p . Strains carrying sltB2 resemble null sltA mutants in being hypersensitive to elevated concentrations of monovalent cations potassium, sodium and lithium, divalent magnesium ion and alkaline media . Preliminary meiotic mapping suggested linkage of sltB to suAadE20 on the left arm of chromosome I.The first evidence of the existence of an additional gene in the SltA-dependent cation tolerance pathway came from a mutation, initially named mx, which arose spontaneously and suppresses the very poor growth of a ne media . Genetic3.2sltB2 is recessive, we used the genomic library based on the auto-replicative pRG3-AMA1 plasmid . Plasmid p4.6 contains a 7958 bp genomic insert which covers a region between coordinates 1,136,528 and 1,143,486 of chromosome I. Plasmid 6.2 carries a fragment of 5031 bp and covers coordinates 1,138,516–1,143,547 of chromosome I . As this deletion might affect flanking genes, strains null only for sltB were generated following standard procedures (sltB transformants were obtained by precisely deleting the coding sequence and replacing it with the pyrG gene from A. fumigatus (AfpyrG) as selectable marker for pyrimidine protrophy. Strain MAD1427 (sltB transformants grew poorly in media containing elevated concentrations of diverse cations or when the pH was adjusted to 8 (sltBΔ strain (MAD3624) were indistinguishable from those shown by the sltB2 strain, and, more interestingly, from those of a null sltA strain  and using strain MAD1427 as recipient. Riboflavin-prototrophic transformants were obtained and have identical phenotypes to those of the AfsltBΔ::pyrG null mutants. Using one of these AfsltBΔ::riboB transformants (MAD3669), we generated by transformation a null sltA allele by replacing the sltA coding region with AfpyrG, as above. Several Af sltAΔ::pyrGAfsltBΔ::riboB strains were obtained. Double mutant strain MAD3814 showed hypersensitive phenotypes to elevated concentrations of monovalent cations , the divalent magnesium cation, and alkalinity, identical with those displayed by single null sltA and sltB strains  and halA24 sltA1 (MAD3114) double mutants. Double mutants sltB2 halA24 (MAD5440) and sltBΔ halAΔ (MAD5442) strain also required calcium supplementation for normal growth and conidiation . In contrast to calcium, magnesium inhibits growth of null sltA and sltB strains  indicated the existence of an alternative splicing of the intron, detected in all species . The founding member of this family of fungal serine proteases is the Ssy5 endoprotease of S. cerevisiae. However the overall sequence conservation between Ssy5 and the C-terminal part of SltB is rather low (25% identity in 68 aa overlap) (A. nidulans protein database (www.aspgd.org) did not detect a putative homologue. Abdel-Sater and collaborators predicted the putative catalytic triad (His–Asp–Ser) of the serine endoprotease Ssy5  was unsuccessful. Searches for possible homologues in fungal genomes allowed us to define conserved residues in this region of SltB. A new HMM domain was identified for the region between residues 1136 and 1194, and searches showed significant similarity to proteins defined as ascomycete specific proteases. Further analyses using MEROPs showed similarity of the SltB 600-1272 moiety to an uncharacterized member of the S64 protease family  from overlap) B. In facase Ssy5  and a siase Ssy5 B. Histidase Ssy5  and Asp-3.5slt− mutations is their ability to suppress the growth-debilitating effect of certain null vps mutations (slt− mutations (vps genes (AfvpsΔ::pyrG) were transformed into pyrG89 recipient strains MAD1427, AMC56 and MAD5131  and lithium cation, only a moderate effect was observed for response to other metal cations  and sltB106 (E743K) beyond their suppression of vpsΔ mutations  changes Thr-972 to Pro and sltB105 substitutes Gly-1156 by Arg, immediately adjacent to the region of Ssy5 similarity , the sltB102 (Y568D) affect a conserved region (see below) between the predicted pseudo-kinase and protease domains.In contrast, mutations affecting a single residue by substitution or deletion were more frequent among utations . sltB104ase Ssy5 . Four otlly null , sltB106milarity . The selhenotype . This mi3.6sltA homologue also contain a sltB homologue and vice versa. This suggests that both proteins are required for pathway function in all Pezizomycotina in which they occur.In order to identify functional domains as well as possible homologues we performed a range of searches using the available genome databases. SltA homologues are present only in species belonging to the Pezizomycotina fungal subphylum  Fig. S4 and thishttp://mobyle.pasteur.fr) using HMM profiles generated from highly conserved regions provided 69 sequences of putative homologues. An alignment between the consensus generated from a multiple alignment and the A. nidulans SltB sequence is shown in sltB coding sequence. sltB affect highly conserved residues among SltB homologues. Also conserved is a short motif identified in the alignment of SltB with the Ssy5p protease  was constructed by gene replacement with the AfriboB gene in the recipient strain MAD2732 , autogenous regulation of sltA expression is possible.However, absence of either SltA or SltB affected transcript levels of the other ′AGGCA3′ ) within g region  stronglyckground . Given t4A. nidulans tolerance to elevated concentrations of monovalent and divalent cations requires the activities of, at least, two zinc-finger transcription factors, CrzA and SltA –Ser1142  predicts the presence of additional elements, not conserved with Ssy5p, modulating the activity of SltB near this putative protease domain. In fact there is no evidence that SltB might be a homologue of Ssy5p, and searches did not find identifiable homologues for the other two elements of the SPS sensor or the cognate transcription factor Stp1. In addition, current data do not indicate a role of SltA or SltB in the utilization of amino acids as nitrogen sources. For example, utilization of serine, threonine and proline are not affected (data not shown). However, the functional relationship between Stp1 and Ssy5 can serve as a working model to understand SltB signaling to SltA, since the membrane-associated transcription factor Stp1 is activated through a proteolysis mediated by Ssy5p  this result suggests that the presence of the various domains in SltB is advantageous to improve viability for vps suppression. In fact, those sltB mutations affecting the region linking the pseudokinase and protease domains are particularly interesting. Four mutations, sltB102, sltB103, sltB106 and sltB57, have been selected and the last two, characterized in more detail, have no effect on cation tolerance, indicating that this connecting region may play a more specific role in interacting with the vps system. Selection and characterization of additional mutations in this region should be informative. An additional interest of selecting further suppressors of vps null alleles is the potential to find new components of the Slt pathway. An attempt to identify new members based on similarities and a pezizomycotina specific distribution has been unsuccessful. The fact that lack of pseudokinase PskA does not affect the cation stress response or suppress vps− mutations clearly demonstrates that a classical forward genetics approach will be needed.Selection of suppressors of the debilitating growth effect of mutations in ESCRT genes has provided an informative collection of mutations in  mutants . Analysi2+/H+ exchanger gene vcxA transcription but positive regulation of sodium ATPase gene enaA (nsdD and steA involved in sexual development and sterigmatocystin biosynthesis has also been described (sltB transcription (sltB transcription start point supports a model in which SltA directly regulates sltB expression. SltA is probably responsible for the elevation of sltB expression under sodium stress. Interestingly, absence of SltB also reduces sltA transcript levels, indicating a strong functional connection between signaling protein and transcription factor. sltA might be subject to autogenous regulation, since consensus target sites for SltA are found in its promoter as tentatively proposed in the scheme in Dual positive and negative function was proposed for SltA, with negative regulation of the vacuolar Caene enaA . A positescribed . Here wecription . The pre


Histopathological prognostication relies on pattern recognition, but as the number of biomarkers increases, human prognostic pattern recognition ability decreases. We have developed an automated quantitative method, hypothesized interaction distribution (HID) analysis, for identifying prognostic patterns of multiple biomarkers in situ. For follicular lymphoma (FL) we have previously demonstrated favourable significance of the transcription factor Yin Yang 1 (YY1) and infiltrating T cells (CD3 +ve) and unfavourable significance of infiltrating macrophages (CD68 +ve).To determine whether the pattern of these biomarkers was important we used HID analysis to determine prognostic significance of their pattern. A tissue microarray (n = 53) was used in triplex immunohistochemistry for YY1, CD3 and CD68, and multispectral imaging used to determine the spatial locations of cells either single, double or triple +ve for YY1, CD3 and CD68. Computer analysis was used to identify doublets of cells within a defined distance of each other such as; YY1/YY1, CD68+/CD68+, YY1&CD68+/YY1&CD3+, and so on for all possible combinations . From this a co-occurrence matrix was derived summarising the relationships of the cells and used in K-nearest neighbour and Kaplan-Meier survival analyses using leave-one-out cross validation. The co-occurrence matrix can was also expressed visually as a heat-map.Two classes with statistically significantly different survival functions (P = 0.025), were identified with median survival of 43 and 112 months. Interestingly, the survival functions appear to be identical until about 30 months, after which they diverge. This indicates that HID analysis may be able to identify patients whose disease will transform. A heat map showed no clear small subset of interactions that appears relate to improved survival, indicating that all the interactions may be important. Linear modelling identified two interactions that may be predictive of survival one of which corresponded to the interaction between pairs of cells positive for the CD3 (P = 0.029), indicating that T cell interaction may be important for survival.An automated method for identifying prognostic patterns in multiply stained tissue sections identified two classes with significantly different outcomes. No single subset of interactions drove this difference, though linear modelling showed that higher levels of T cell interaction were associated with a favourable outcome. This is in line with previous studies, though was identified using the HID method automatically without any a priori knowledge and without expert histopathological input. The method can be extended to higher order multiplex analysis, enabling identification of prognostic patterns of multiple biomarkers.


Our result highlighted that the gross metabolic changes observed in G12C KRAS mutant cells growing in culture were also maintained in the derived xenograft model, suggesting that a simple in vitro cell model can give important insights into the metabolic alterations induced by cancer. This is of relevance for guiding effective targeting of those metabolic traits that underlie tumor progression and anticancer treatment responses.Oncogenes induce metabolic reprogramming on cancer cells. Recently, G12C KRAS mutation in isogenic NSCLC cell line has been shown to be a key player in promoting metabolic rewiring mainly through the regulation of glutamine metabolism to fuel growth and proliferation. Even though cell lines possessing many of the genetic backgrounds of the primary cancer they derive from could be a valuable pre-clinical model, they do not have the additional complexity present in the whole tumor that impact metabolism. This preliminary study is aimed to explore how cancer cell metabolism in culture might recapitulate the metabolic alterations present  In particular it has been demonstrated that KRAS activation supports the decoupling of glycolysis and TCA metabolism, with glutamine supplying increased carbon to drive the TCA cycle in different cancer cell lines such as NSCLC, pancreatic and colon123in vivo invariably introduces new complexities, but has, on the other hand, the great advantage of taking into consideration how the whole tumor heterogeneity and the interaction between different cell types as well as nutrient availability impact on its metabolismCell culture studies have revealed how different oncogenic mutations and nutrients impact on cancer metabolism. Studies using in vivo.So far limited data exist that address how faithfully cancer metabolism in culture recapitulate the metabolic alterations observed in cancer models in vitro could be translated into the metabolism of the whole tumor.In this explorative investigation we applied a quantitative metabolomics profiling to human NSCLC isogenic cell lines harboring G12C or WT KRAS isoforms and to their derived tumors implanted in immunodeficent mice, in order to decipher whether and how much the cancer cell metabolic findings obtained in-vitro culture systems, we investigated the KRAS associated metabolomic signatures on NSCLC cell lines harboring G12C mutation or WT KRAS both in monolayer culture and in corresponding tumors derived NSCLC xenograft models.With the aim of better understanding how whole-animal physiology impacts on metabolic alteration observed in in vitroin vivo.The clones had similar growth rate in vitro in cells growing in monolayer  as well as on their derived tumor xenograft models  from which metabolite extracts for each condition were analysed.On the basis of the results obtained in our recent publicationConcentrations of metabolites passing our filtering criteria in cell systems and xenograft model are listed as µM in To gain a deeper understanding of the capability to translate our cell monolayer metabolic fingerprint to the biochemistry of whole tumor, an orthogonal projection to latent structures-discriminant analysis (OPLS-DA) was used. OPLS-DA models demonstrated robust group separation between G12C and WT KRAS isoforms both in cell system  and xenograft model  as shown in in vivo. Particularly, protein biosynthesis, ammonia recycling and urea cycle were the most enriched pathways both in G12C KRAS mutant cell system and xenograft model  down regulation of low-unsaturated very long-chain phosphatidylcholine (PC) species in comparison to WT KRAS C. This l10in vitro cell model can give important insights into the metabolic alterations induced by cancer cells to fuel their proliferation and survival. Our data also indicate that the gross changes observed in vitro are maintained in a more complex system of cancer growth in vivo. This implies that pharmacological modulation of cancer cell metabolism can be studied both in vitro and in vivo thus allowing a proper evaluation of the impact of new molecules acting at different steps in the cell metabolism. The possibility to evaluate the pharmacological  activity of new molecules and combination and to correlate their activity with the pharmacodynamic changes observed at metabolic level, will definitely help in defining the best rational way of use and combine these new molecules with potential activity as anticancer agents.Altogether, the data of our pilot investigation highlighted that key metabolic pathways involved in the metabolic rewiring induced by G12C KRAS mutation in cells growing in culture were also maintained in the derived xenograft model, suggesting that a more simple in vitro to in vivo system due to tumor heterogeneity, tumor environment and KRAS mutational status able to affect metabolism as reported by two recent articles12Nevertheless, caution has to be taken when translating tumor metabolism from 2 in air1Human non-small-cell lung carcinoma NCI-H1299 overexpressing G12C and WT KRAS isoforms were grown in RPMI1640 medium with 500 µG/mL of G418 (Gibco) added. G12C and WT KRAS clones generation and characterization are described in our previous articles6Immunodeficient mice were obtained from Harlan-Italy and maintained under specific pathogen-free conditions with food and water provided ad libitum. Procedures involving animals and their care were conducted in conformity with the following laws, regulations, and policies governing the care and use of laboratory animals: Italian Governing Law , Mario Negri Institutional Regulations and Policies providing internal authorisation for persons conducting animal experiments , the NIH Guide for the Care and Use of Laboratory Animals (2011 edition), the EU directives and guidelines (EEC Council Directive 2010/63/UE) and in line with Guidelines for the welfare and use of animals in cancer research. Animal experiments has been reviewed and approved by the IRFMN Animal Care and Use Committee (IACUC) that includes members “ad hoc” for ethical issues. Animals were housed in the Institute’s Animal Care Facilities, which meet international standards; they are regularly checked by a certified veterinarian who is responsible for health monitoring, animal welfare supervision, experimental protocols and procedures revision.6 cells/mouse) in the flank of immunodeficient mice (Harlan Laboratories). When the tumor reached approximately 500 mm3 as determined by caliper measurement of its diameters, tumors from 8 mice (4 bearing wt KRAS and 4 bearing G12C KRAS) were excised, and immediately frozen in liquid nitrogen and stored under cryogenic conditions until analysis. A portion of the tumor was used to determine the percentage of normal infiltrating cells (of murine origin) in the tumor (human origin) by species specific Real-Time PCR measurement of DNA.Exponentially growing cells (both WT KRAS and G12C KRAS) were implanted subcutaneously (5 × 10Genomic DNA extracted with Maxwell 16 (Promega) was amplified by 7900HT Sequence Detection System (Life Technologies) with Primers and TaqMan probes specific for human and murine actin purchased as ready-to-use solutions (Life Technologies).NCI-H1299 cell lines harbouring G12C or WT KRAS isoforms were seeded at 75000 cells/mL in Petri dishes in a total volume of 10 mL in biological replicates (n = 9) for each clone. Forty-eight hours after seeding, all clones were in exponential growth with same proliferation rate. Metabolites were quenched and extracted as reportedFrozen tumor tissue samples from each of the xenograft models (n = 4 for each clone) were disintegrated with a Mikro-Dismembrator S  at 3000 revolutions per minute for 40 s. The obtained powder was resuspended in ice-cold MeOH (3 µL/mG of tumor tissue) and homogenized for 1 min. Homogenized samples were subsequently centrifuged for 15 min at 10000 × g and supernatants stored at −80 °C. Thirty µL of each supernatant were used for targeted metabolomics analysis.L) using an LC/MS  method  followed by FIA-MS . For analytical specifications, refer to the AbsoluteIDQ p180 Kit manuals and A targeted quantitative approach using a combined direct flow injection and liquid chromatography (LC) tandem mass spectrometry (MS/MS) assay  was applied for the metabolomics analysis. The assay quantifies 188 metabolites from five analyte groups: acylcarnitines, amino acids, biogenic amines, hexoses (sum of hexoses), PCs, and sphingomyelins (SMs) . The metMetabolites concentrations (µM) from each experimental condition (cell clones and tumor tissues) were submitted to the SIMCA-P13 software package (Umetrics) for multivariate data analysis. Metabolite levels were Pareto scaled with mean centering and analyzed by orthogonal partial least-squares discriminant analysis (OPLS-DA) to maximize class discrimination. Loading S-plot generated by OPLS-DA were used to visualize the relationship between covariance and correlation within the OPLS-DA results. For the extraction of metabolites with highest magnitude and highest reliability in discriminating between groups, the cut-off of variable confidence was set at 0.05 for the increased metabolites and −0.05 for the decreased metabolites in the S-plot. The selected metabolites were then submitted to pathway analysis. Mann-Whitney-Wilcoxon test  was performed on the discriminant metabolites set to identify those whose concentration significantly (p < 0.05) changed between G12C and WT KRAS isoforms in cell and xenograft models.For biological interpretation of the metabolite dataset, we mapped the quantified metabolites to the KEGG pathway database , using MetaboAnalyst 3.0, a comprehensive online tool suite for metabolomic data analysis and interpretation . Enrichment analysis (EA) tools were used to identify metabolic pathways that were most likely to be associated with the given list of the discriminant metabolites derived from the S-plots analysis.How to cite this article: Brunelli, L. et al. Comparative metabolomics profiling of isogenic KRAS wild type and mutant NSCLC cells in vitro and in vivo. Sci. Rep.6, 28398; doi: 10.1038/srep28398 (2016).


MSLN) gene encodes a 71 kilodalton (kDa) precursor protein that is processed into megakaryocytic potentiating factor (MPF), a 31 kDa protein that is secreted from the cell, and mature mesothelin (mMSLN), a 40 kDa cell surface protein. The mMSLN binds to CA125, an interaction that has been implicated in the intra-cavitary spread of mesothelioma and ovarian cancer. To better define the role of MPF and mMSLN, growth of the lung cancer cell line A549 was evaluated in immuno-deficient mice with inactivation of the Msln gene. We observed that Msln–/– mice xenografted with intraperitoneal A549 tumors survive significantly long than tumor-bearing Msln+/+ mice. When tumor-bearing Msln–/– mice are supplemented with recombinant MPF (and to a lesser extent mMSLN), most of this survival advantage is lost. These studies demonstrate that MPF and mMSLN have an important role in the growth of lung cancer cells invivo and raise the possibility that inactivation of MPF may be a useful treatment for lung and other MSLN expressing cancers.The mesothelin ( It is also highly expressed in epitheliod mesotheliomas, which are derived from normal mesothelial cells, and aberrantly in many other cancers including ovarian, pancreatic, lung, stomach, cholangiocarcinoma and triple negative breast cancer MSLN gene encodes a 71 kDa precursor protein that is processed into two mature proteins: Megakaryocytic Potentiating Factor (MPF) and mature MSLN (mMSLN). MPF is a 31 kDa glycoprotein that is secreted from the cell into the blood. The mMSLN is a 40 kDa glycoprotein protein that remains bound to the cell membrane by phosphatidyl inositol Msln gene knock out(–/–) mice indicate that the gene is not essential for normal development and reproduction, but several recent studies have raised the possibility that MSLN might regulate cancer cell growth MSLN gene MSLN has been implicated in significant enhancement of tumor cell growth and migration invitroThe mesothelin Msln gene is inactivated, preventing the production of mMsln and Mpf protein. These mice reproduce normally and no abnormalities in their growth or other properties have been observed. Because these mice do not make Mpf and do not have Msln on the cells that line the peritoneal cavity, we have used them to investigate whether the absence of Msln affects the growth of lung cancer cells inoculated within the peritoneal cavity of these mice.We have generated athymic nude mice in which the The lung cancer cell line A549 was obtained from Dr. Hisataka Kobayashi  and its identity was confirmed by DNA fingerprinting using Short Tandem Repeats assay. The human epidermoid carcinoma cell line A431 was purchased from ATCC  and maintained in the laboratory as recommended. Immunohistochemical staining for MSLN and CA125 was performed by Histoserv, Inc  using mouse anti-MSLN 5B2  as previously described Msln–/– mice obtained from NCI-Frederick were housed in micro-isolator cages throughout the course of the experiment. All mice used for experiments were females, weight (15–20 g) and age (7–10 weeks) matched. All injections were intraperitoneal (IP) unless otherwise indicated. A549 and A431 IP xenografts were established by inoculating 2×106 cells in 200 µl of cell suspension in phosphate buffered saline (PBS). For the subcutaneous model, 2×106 A549 cells were injected into the right thigh. All recombinant protein treatments were performed by IP injection of 25 µg study protein in 200 µl PBS, three times weekly for a total of three weeks. For survival studies, mice were monitored daily and sacrificed by CO2 inhalation when moribund.The Animal Study Proposals (LMB-053 and LMB-014) were approved by the NIH Animal Care and Use Committee. All animal experiments including treatment and sacrifice were conducted according to the institutional guidelines of the NIH. Athymic nude mice (NCr-nu/nu) and Nude/Msln null mice in C57/Bl6 genetic background (Msln–/–) Msln heterozygous allele. Male heterozygote mice from each generation were used to back cross with WT BALB/c females and back crossing was performed for more than ten generations. Males heterozygous for Msln in BALB/c genetic background were crossed with athymic nude (Fox N) females to generate Fox N null/Msln–/– mice. Since Fox N null/Msln–/– female mice are not good breeders, we used Msln–/–/Fox N Het females as the breeding partners for Fox N null/Msln–/– males to maintain and generate Fox N null/Msln–/– females for our experiments.Male To generate recombinant versions of mMSLN and MPF, mMSLN-rFc and MPF-rFc were expressed as fusion proteins with rabbit IgG (rFc) in HEK293T cells and purified as previously described Cells were harvested, washed and re-suspended in ice-cold FACS buffer (PBS with 5% FBS and 0.1% sodium azide), then incubated with anti-mesothelin antibody, MN  or Morab-009  or anti-MUC16 antibody, OC125  at concentration of 5 µg/ml. Isotope-matched antibody was used as a control. The binding of primary antibodies to tumor cells was detected by using either goat anti-mouse conjugated with R-phycoerythrin (R-PE) (Invitrogen) or with goat anti-mouse conjugated with FITC (Biosource). The fluorescence associated with the live cells was determined using a FACS Calibur (BD Biosicences). Geometric means were expressed as mean fluorescence intensity (MFI). The MFI of cells was compared with the MFI from a standard curve of R-PE-conjugated calibration beads  and the number of mMSLN sites per cell was estimated. Results were similar with MN (n = 2 experiments) or Morab-009 (n = 3 experiments).A549 cells stably transfected with either vector control (A549/pcDNA) or MPF expression vector (A549/MPF) were plated into dishes containing equal amount of complete medium and incubated for 48 hours. Total cell population was counted using a Cellometer Vision cell counter  An equivolume aliquot of conditioned medium was removed from each dish and the concentration of MPF in the solution was quantified using a proprietary human MPF enzyme-linked immunosorbent assay kit .3 cells/well), and incubated for 15–21 days at 37°C. After Crystal Violet staining the plates were washed for 1 hour then scanned to produce digital images. Colonies were counted within a pre-specified, centrally-placed square field with side length equal to one-third of the well diameter. Multiple wells (n) were counted for each of three independent experiments. The mean number of colonies for each experiment are reported with standard deviation (SD). KB cells were used as a positive control for colony formation.A549/pcDNA or A549/MPF stable transfectants were suspended in 0.35% agar, plated in 6 well plates  for Mac (GraphPad software).Msln–/– mice. To ensure that the Msln gene was inactivated and that the mice did not make Msln we first confirmed the deletion using polymerase chain reaction to screen for the deleted alleles of the Msln gene (data not shown). Lack of mMsln protein expression was confirmed by immunohistochemical analysis of mouse tissues using rabbit polyclonal anti-Msln antibody followed by color detection as previously described 3 mMSLN binding sites/cell) but robust expression of CA125 . Nearly 25% of the Msln–/– mice survived over four months  mice. Therefore, we synthesized recombinant MPF-rFc, mMSLN-rFc, and a control CD22-rFc protein. The strategic addition of the rabbit Fc moiety significantly increases the half-life of these molecules, but is not expected to interfere with function. A549 cells were injected into the peritoneal cavities of Msln–/– mice as before, then, beginning on the day of implantation, the mice were treated with one of the recombinant Fc proteins every other day for a total of six doses. As summarized in Msln–/– mice treated with the CD22-rFc control peptide remained 46 days. However, median survival of mice treated with MPF-rFc decreased to 31 days, similar to the survival of Msln(+/+) mice injected with A549 cells . We noted no obvious difference in the growth rate or morphology of the MPF-transfected A549 cells when compared with the vector-transfected control (data not shown). Anchorage-independent growth of these cells in soft agar was also assessed. As shown in MPF-transfected A549 cells and the vector-transfected control line. These experiments suggest that MPF does not directly promote the growth of A549 cells.To determine if MPF acts directly on the A549 cells to enhance their growth or aggressiveness, we stably transfected the Msln gene and is permissive to the growth of human cancer cells. The A549 lung cancer cell line used to form IP tumors in our experiments makes only minimal amounts of mMSLN, such that all MPF and mMSLN must be supplied exogenously. We observed a statistically significant 15 day difference in survival between Msln+/+ and Msln–/– mice bearing intraperitoneal A549 tumors. This finding was cell-line specific and, interestingly, not observed in a subcutaneous model. These results indicate that a product of the Msln gene plays an important role in promoting invivo tumor growth and progression for some types of cancer cells growing within the peritoneal cavity.For our study, we created a mouse model that lacks the MSLN gene. Both proteins are highly expressed by a number of solid tumor malignancies including mesothelioma, pancreatic and ovarian cancers MSLN gene can alter tumor growth, progression or invasiveness to produce a more aggressive tumor phenotype. Overexpression of the MSLN gene was shown to enhance interleukin (IL)-6 signaling MSLN gene in a human breast cancer cell line promoted cell survival and anchorage-independent growth invitroMSLN gene in regulating growth and apoptosis via both p53-dependent and independent pathways in pancreatic cancer cells invitro studies, the pro-tumorigenic effects of MSLN gene manipulation were presumed to be mediated by changes in expression of mMSLN, although the genetic manipulations in the experiments should produce similar alterations of MPF levels.MPF and mMSLN are both protein products made by expression of the Msln–/– mice. We found that MPF supplementation decreased animal survival 15 days compared to mice treated with a control protein. While supplementation with mMSLN did decrease survival a statistically significant 4 days, it is clear that MPF is the more potent factor in our model.To separate contributions made by MPF or mMSLN to the phenotype we observed in our study, we supplied exogenous recombinant protein to the invivo, forced overexpression of MPF produced no growth advantage invitro. This suggests that the effect of MPF on tumor aggressiveness is not mediated by a direct action of MPF on the tumor, but perhaps by working indirectly on other cells. Although it might be tempting to consider that MPF may have an immune effector function, our experiments were conducted in lymphocyte-deficient mice, suggesting that another mechanism is more likely to be responsible. Identification of the cell type(s) responsible for mediating this effect of MPF is beyond the scope of the current study.MPF was originally identified as a growth factor that could promote colony formation of megakaryocytes when given in combination with IL-3 Although our observations were made using a single cell type, the effect we observed on survival was profound and highly reproducible. Our data suggest that MPF could be an important mediator of tumor aggressiveness in the peritoneal cavity, and that inhibition or sequestration of MPF might be clinically useful in slowing the growth of some cancers that grow in the peritoneal cavity. Studies to test this hypothesis have been initiated.


Crohn's disease (CD) is a chronic inflammatory disease of the gastrointestinal tract. Genetic polymorphisms can confer CD risk and influence disease phenotype. Indoleamine 2,3 dioxygenase-1 (IDO1) is one of the most over-expressed genes in CD and mediates potent anti-inflammatory effects via tryptophan metabolism along the kynurenine pathway. We aimed to determine whether non-synonymous polymorphisms in IDO1 or IDO2  are important either as CD risk alleles or as modifiers of CD phenotype.Utilizing a prospectively collected database, clinically phenotyped CD patients (n = 734) and non-IBD controls (n = 354) were genotyped for established IDO1 and IDO2 non-synonymous single nucleotide polymorphisms (SNPs) and novel genetic variants elucidated in the literature. Allelic frequencies between CD and non-IBD controls were compared. Genotype-phenotype analysis was conducted. IDO1 enzyme activity was assessed by calculating the serum kynurenine to tryptophan ratio (K/T).IDO1 SNPs were rare  and not linked to Crohn's disease diagnosis in this population. IDO1 SNPs did however associate with a severe clinical course, presence of perianal disease, extraintestinal manifestations and a reduced serum K/T ratio during active disease suggesting lower IDO1 function. IDO2 minor allele variants were common and one of them, rs45003083, associated with reduced risk of Crohn's disease (p = 0.025). No IDO2 SNPs associated with a particular Crohn's disease clinical phenotype.This work highlights the functional importance of IDO enzymes in human Crohn's disease and establishes relative rates of IDO genetic variants in a US population. Crohn's disease (CD) is a chronic inflammatory bowel disease (IBD) affecting of millions of people of all races worldwide. Current evidence suggests that CD occurs in genetically susceptible individuals who develop loss of tolerance and a resultant chronic immune response against commensal luminal microbiota, likely in response to an antecedent environment trigger. Indoleamine 2,3 dioxygenase-1 (IDO1) is a widely expressed enzyme which is the initial and rate limiting step of tryptophan catabolism along the kynurenine pathway. IDO1 expression is inducible by inflammatory stimuli including cytokines and toll like receptor agonists. The resulting suppression of local tryptophan and increase in bioactive kynurenine pathway metabolites functions to reduce inflammation and promote immune tolerance via several mechanisms. Based on this experimental and observed human data, we hypothesized that individuals carrying a functionally relevant SNP of the IDO1 gene may exhibit unchecked inflammation and thus experience a more severe disease course if affected by Crohn's. Though not identified as such in GWAS studies to date, it is also possible that IDO1 SNPs may confer risk for development of CD in some populations. To address these hypotheses we examined a prospectively enrolled cohort of well-characterized CD patients and a non-IBD control cohort for known IDO1 SNPs. We also examined the same population for the variants of the more recently discovered gene analog of IDO1, IDO2. While its expression is more restricted than that of IDO1, its expression in the colon is reported. This protocol was approved by the Human Research Protection Office of Washington University School of Medicine and all clinical investigation was conducted according to the principles expressed in the Declaration of Helsinki. All participants provided their written informed consent to participate in this study. To identify nonsynonymous single nucleotide variants for IDO1 and IDO2 and their expected frequencies we used the online public databases HapMap All patients included in this study were prospectively enrolled by providing written informed consent as part of the Washington University in St Louis Division of Gastroenterology's Digestive Disease Research Cores Center (DDRCC) BioBank core. This repository included blood, saliva, and/or tissues for genotyping, obtained via recruitment in consecutive fashion during inpatient and outpatient visits as previously described. Primer specific genotyping was performed by Sequenom MassARRAY  as previously described by our group. The kynurenine to tryptophan (K/T) ratio is a reliable method to estimate IDO enzymatic activity which controls for variations in dietary tryptophan intake. http://www.r-project.org). Mann-Whitney U test was used for non-parametric continuous variables  and Software R , included more males , and had fewer African Americans  in comparison to the non-IBD group. These differences likely reflect the IBD patient population compared to the cohort of individuals that volunteer to participate in open access studies or who are eligible for colorectal cancer screening in the study enrollment region.A total of eight patients in the CD cohort carried IDO1 genetic variants compared to six individuals in the non-IBD cohort . Predicted and experimental IDO1 minor allele frequencies are shown in We next sought to determine if there were any potential differences in the prevalence of IDO2, a gene paralog to IDO1, variants between a CD and non-IBD population. Observed variant minor allele frequencies of IDO2 were common, but fell within the predicted minor allele frequencies . We did Several patients carried overlapping IDO2 minor allele SNPs. When considering all gene variants, the likelihood of carrying at least one IDO2 SNP was similar between cohorts . Moreover, the number of those who were homozygous for at least one IDO2 SNP was not different between non-IBD and CD patients .There is strong clinical and preclinical evidence implicating a role for IDO1 in modulating IBD disease activity. To validate the multivariate comparisons, we performed univariate analysis and found extraintestinal disease manifestations (EIM) including IBD-associated arthritis and uveitis were more common in IDO1 SNP patients . Perianal involvement, an indicator of disease severity, also more commonly affected the SNP carrying cohort . While race could be considered an influential factor in these phenotypes, both results remain significant on univariate analysis when comparing within African Americans only (see table footnote).Individual patient characteristics are described in We further sought to examine whether IDO1 SNPs were associated with detectable differences in enzyme function in vivo. All patients with IDO1 SNPs had serum sampled during a period of moderate to severe Crohn's disease activity. Serum tryptophan and kynurenine levels were determined using an established method and compared to CD patients without IDO1 variants during a time of similar disease activity. IDO2 variants were more common among White than African Americans. Penetrating disease was less common in patients with any homozygous IDO2 SNP on univariate analysis. However, this finding did not stay significant on multiple testing and no other clinical characteristics were identified to be different when comparing patients with no identified SNPs to those who were either heterozygous or homozygous for IDO2 SNPs . The samUtilizing a prospectively collected database of clinically phenotyped IBD patients, we genotyped for established IDO1 and IDO2 SNPs and novel genetic variants elucidated in the literature. IDO1 SNPs were rare and not linked to Crohn's disease risk in this population. However, they were associated with a severe clinical course, presence of perianal disease, extraintestinal manifestations and a reduced serum K/T ratio during active disease. IDO2 SNPs were common and varied in frequency according to race and CD risk. No IDO2 SNPs associated with a particular Crohn's disease clinical phenotype. This work highlights the functional importance of IDO enzymes in human Crohn's disease and establishes relative rates of IDO genetic variants in a large US population.The finding that one of the IDO2 minor allele variants conferred CD risk is provocative, particularly given that this SNP, rs10109853 (R248W), confers a 90% reduction in enzymatic activity. Extra-intestinal manifestations and perianal disease were both more common in IDO1 SNP carrying patients. Though overlap exists between the risk alleles for IBD and other inflammatory diseases, very little is known as to whether any of these polymorphisms confer risk for IBD associated extra-intestinal manifestations. The finding that IBD associated arthritis and uveitis occurred in patients with IDO1 SNPs is supported by studies linking elevated IDO1 expression to disease activity and pathogenesis in both inflammatory arthritis The impact of IDO1 polymorphisms on enzymatic activity was assessed using the K/T ratio, a well-established surrogate for IDO1 function. We found that in composite, non-synonymous IDO1 SNPs conferred a diminishment in enzymatic activity (mean K/T of 42.1 vs 86.9 for non-IDO1 SNP CD patients). This average was numerically higher, though not statistically different from the K/T of 35.1 in a non-CD control cohort of patients examined previously by our group. Sample size is a significant study limitation especially in comparison to larger genome wide association studies. As IDO1 SNPs are rare, a larger cohort would be required to assess the association of disease risk. Using a case:control ratio of 1∶5, the determination of a 50% increase in risk could be achieved by including between 552 and 2985 cases , assuming 80% power and Type 1 error rate of 0.05. In summary, our data newly link IDO SNPs and minor allele variants to Crohn's disease risk and phenotype. As IDO1 variants are modestly rare, large disease and control cohorts will need to be evaluated to identify if these SNPs confer increased disease risk or associate with less common disease phenotypes. Moreover, confirmative studies should be extended to include an ulcerative colitis cohort. Finally, if therapies directed at enhancing or blocking IDO function move from early phase


We sought to address the significance of isolated follicles that exhibit atypical morphologic features that may be mistaken for lymphoma in a background of reactive lymphoid tissue. Seven cases that demonstrated centroblast-predominant isolated follicles and absent BCL2 staining in otherwise-normal lymph nodes were studied. Four of seven cases showed clonal B-cell proliferations amid a polyclonal B cell background; all cases lacked the IGH-BCL2 translocation and BCL2 protein expression. Although three patients had invasive breast carcinoma at other sites, none were associated with systemic lymphoma up to 44 months after diagnosis. The immunoarchitectural features of these highly unusual cases raise the question of whether a predominance of centroblasts and/or absence of BCL2 expression could represent a precursor lesion or atypical reactive phenomenon. Differentiating such cases from follicular lymphoma or another mimic is critical, lest patients with indolent proliferations be exposed to unnecessarily aggressive treatment. In recent years, recognition of aberrant strong coexpression of the germinal center marker CD10 and the anti-apoptotic protein BCL2, has been described within isolated secondary follicles of otherwise-normal lymph nodes.[eoplasms.IGH-BCL2 gene rearrangements in the peripheral blood and tissues of patients with no evidence of FL.[The clinical significance of FLIS is still of uncertain significance, particularly because numerous studies have reported the presence of non-neoplastic cells that harbor ce of FL.–20 Diagnce of FL.–23 To dace of FL., 21IGH@ gene rearrangements were performed. The diagnostic challenges and clinical implications of these unusual findings are described.Herein, we report seven cases characterized by isolated abnormal follicles composed of atypical centroblasts within otherwise-normal lymph nodes that show significant differences from previously described cases. Detailed morphologic and immunophenotypic characterization was undertaken, and in cases with sufficient tissue, cytogenetics and molecular studies for clonal A search of the Department of Pathology database at Stanford University Medical Center to include the terms “atypical follicles”, "follicular lymphoma in situ," and "follicular lymphoma-like B-cells", yielded cases received between 2010 and 2014. These showed either an abnormal pattern of strong CD10 and BCL2 coexpression within otherwise-normal secondary follicles or isolated abnormal follicles composed predominantly of centroblasts within lymph nodes with preserved architecture. The latter group of cases was selected for inclusion. Institutional Review Board approval was obtained from Stanford University for these studies. Clinical information including follow-up in the form of subsequent lymph node samples, staging bone marrow biopsies, imaging studies and clinical evaluations was recorded. Patient records and information were anonymized and de-identified prior to analysis.The morphologic characteristics of each case were recorded in a manner similar to reports of follicular lymphoma in situ, includinImmunohistochemical stains were performed on 4-micron thick, formalin-fixed, paraffin-embedded whole tissue sections using automated staining platforms . Primary antibodies and conditions used for immunohistochemistry are detailed in BCL2, IRF4 and BCL6 gene rearrangements by fluorescence in situ hybridization using a 5’/3’ breakapart probes for the BCL2 gene , IRF4 gene  and the BCL6 gene (ZytoVision). Briefly, using a Vysis VP2000™ slide pretreatment instrument and reagents , slides were deparaffinized with CitroSolv™, digested with a 10% pepsin solution at 37°C, pre-treated with a sodium thiocyanate solution at 80°C, re-fixed in 10% buffered formalin, and dehydrated in an ethanol series. Dried, dehydrated slides were mounted with probe solution per manufacturer’s instructions, denatured with a Vysis® HYBrite instrument at 80°C for six minutes and hybridized for 48 hours at 37°C. Slides were washed with 2xSSC/0.3% NP-40 at 73°C for two minutes, counterstained with DAPI and analyzed with an Olympus BX51 microscope equipped with an 100x oil immersion objective, appropriate fluorescence filters and CytoVision® imaging software .Four micron formalin-fixed, paraffin-embedded tissue sections were analyzed for IGH@ gene rearrangements was performed on DNA obtained from paraffin-embedded tissue samples in a subset of cases using the IGH Gene Rearrangement Assay  according to the manufacturer’s instructions. Three cases  in which unstained sections were available were further evaluated by microdissection. To accomplish this, unstained sections were overlaid on H&E stained sections and the area of interest marked and removed by scraping. Desired portions were extracted using proteinase K digestion followed by QIAgen DNA column purification . Elution volume of 22uL with heat and shaking were used with a yield of 19uL. Clonality was assessed using Invivoscribe B-clonality kit, primer mix A (FR1-JH) and primer mix F (amplification control). Tube F was used to assess amplifiability and to estimate yield. No spectrophotometric assessment of yield was performed due to very low amount of sample available for these assays. Uninvolved nodes were subjected to clonality assessment in parallel. All except the uninvolved sample of case 5, among the three paired samples yielded amplifiable DNA. Analyses of amplicons were performed by fragment analysis using capillary electrophoresis on the ABI 3100 Genetic Analyzer .Polymerase chain reaction (PCR) for clonal The seven patients in this cohort included five women aged from 51 to 61 years old and two men aged six and 61. Anatomic sites of involvement included peripheral as well as internal sites such as periaortic lymph node and rectal mucosa-associated lymphoid tissue. Six cases were sampled during evaluation for unrelated medical conditions  and seventh was a cervical lymph node removed for lymphadenopathy. Although three of the patients had invasive ductal carcinoma of the breast, none of the patients had associated follicular lymphoma (or other subtypes of lymphoma), detected either in the same lymph node or systemically at the time of biopsy. Bulky lymphadenopathy was not detected in any of the patients in our cohort. A summary of clinical features and follow-up is presented in The seven cases in our series showed scattered follicles composed almost exclusively of centroblasts localized to isolated follicles in lymph nodes with preserved normal architecture . MorpholCytologically, the centroblasts were enlarged with vesicular chromatin and prominent nucleoli. They demonstrated nuclear membrane irregularity and deviated from the morphology seen in the surrounding normal lymph node. Furthermore, atypical mitoses were appreciated with admixed tingible body macrophages but lacking centrocytes. Overall, these features, in the context of follicular lymphoma, would warrant a grade 3B designation; however, they were not assigned a grade in the current clinical context.in situ described thus far  demonstrated altered expression of germinal center B-cell marker, HGAL, which show overexpression of the protein throughout the affected follicle. In comparison, the surrounding reactive follicles show a gradient of HGAL staining with higher intensity in dark zones, which matches the polarization of normal germinal centers .BCL2 gene rearrangement. None were positive, which is in keeping with over 50 percent of grade 3 follicular lymphomas lacking the IGH-BCL2 rearrangement. Two cases were further tested by FISH for BCL6 and IRF4 gene rearrangements  and were found to be negative  translocation involving the rk of FL. Up to 10rk of FL.t/IGH-BCL2 translocation-positive cases that lacked BCL2 reactivity in cases of FL associated with FLIS.[BCL2 breakpoint region in the FL component of a case with simultaneous FL and FLIS.[Jegalian and colleagues also reported t4;18/IGH-and FLIS.These finand FLIS.–33 TheseIGH-BCL2 translocation. The seven cases we describe are distinctly different from the previously reported cases in that they demonstrate a predominance of centroblasts and lack expression of BCL2.FLIS is recognized as a follicular proliferation of monoclonal BCL2+ CD10+ lymphoid cells within scattered germinal centers of a lymph node with otherwise intact architecture. Cases described to date have been comprised of predominantly centrocytes and contained the While the clinical significance of FLIS will depend largely on the results of long-term follow-up, initial studies suggest progression to follicular lymphoma is an infrequent event. It may bEarly detection of an aggressive B-cell lymphoma or carcinoma may lead to immediate intervention. Our cases showed involvement of rare scattered follicles in an architecturally intact lymph node, but contained increased centroblasts histologically consistent with grade 3B FL. Just as in typical FLIS, these cases were incidental findings in lymph nodes removed for various reasons and the patients did not show clinical or radiological evidence of lymphoma at the time of presentation. It is critical to recognize such cases, both for further study, and to potentially avoid overly aggressive treatment of an indolent lesion.