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Ulcerative colitis (UC) and Crohn's disease (CD) are common inflammatory bowel diseases producing intestinal inflammation and tissue damage. Although emerging evidence suggests these diseases are distinct, approximately 10% of patients remain classified as indeterminate inflammatory bowel disease even after invasive colonoscopy intended for diagnosis. A molecular diagnostic assay using a clinically accessible tissue would greatly assist in the classification of these diseases. In the present study we assessed transcriptional profiles in peripheral blood mononuclear cells from 42 healthy individuals, 59 CD patients, and 26 UC patients by hybridization to microarrays interrogating more than 22,000 sequences. Supervised analysis identified a set of 12 genes that distinguished UC and CD patient samples with high accuracy. The alterations in transcript levels observed by microarray were verified by real-time polymerase chain reaction. The results suggest that a peripheral blood mononuclear cell-based gene expression signature can provide a molecular biomarker that can complement the standard diagnosis of UC and CD.

The JAK2 V617F mutation identifies a subgroup of MDS patients with isolated deletion 5q and a proliferative bone marrow

Given their accessibility, surrogate tissues, such as peripheral blood mononuclear cells (PBMC), may provide potential predictive biomarkers in clinical pharmacogenomic studies. In leukemias and lymphomas, the prognostic value of peripheral blast expression profiles is clear; however, it is unclear whether circulating mononuclear cells of patients with solid tumors might yield profiles with similar prognostic associations.In this study, we evaluated the association of expression profiles in PBMCs with clinical outcomes in patients with advanced renal cell cancer. Transcriptional patterns in PBMCs of 45 renal cell cancer patients were compared with clinical outcome data at the conclusion of a phase II study of the mTOR kinase inhibitor CCI-779 to determine whether pretreatment transcriptional patterns in PBMCs were correlated with eventual patient outcomes.Unsupervised hierarchical clustering of the PBMC profiles using all expressed genes identified clusters of patients with significant differences in survival. Cox proportional hazards modeling showed that the expression levels of many PBMC transcripts were predictors for the patient outcomes of time to progression and overall survival (time to death). Supervised class prediction approaches identified multivariate expression patterns in PBMCs capable of assigning favorable outcomes of time to death and time to progression in a test set of renal cancer patients, with overall performance accuracies of 72% and 85%, respectively.The present study provides the first example of gene expression profiling in peripheral blood, a clinically accessible surrogate tissue, for identifying patterns of gene expression associated with higher likelihoods of positive outcome in patients with a solid tumor.

Expression profiling has demonstrated that transcriptomes of primary malignancies differ from those in normal tissue. It is unknown, however, whether there exist "surrogate" transcriptional markers in peripheral blood mononuclear cells (PBMCs) of patients with solid tumors. We identified transcripts expressed differentially between PBMCs from renal cell carcinoma patients and normal subjects, some of which appear to reflect specific immune responses of circulating cells. We also identified small sets of predictor genes distinguishing PBMCs from renal cell carcinoma patients and normal volunteers with high accuracy. The present findings have important implications for diagnosis and future clinical pharmacogenomic studies of antitumor therapies.

Ewing's sarcoma affects mostly adolescents and young adults with tumors that predominate in bone, characterized by the presence of fusion proteins created by chromosomal translocations (EWS-FLI1, EWS-ERG etc.). Recent studies demonstrated an interaction between EWS fusion proteins and PARP1; increased DNA damage and elevated PARP levels upon fusion protein expression as well as sensitivity to PARP inhibition have been reported. E7449, a potent, orally available PARP inhibitor was evaluated in various xenograft models of Ewing's sarcoma as a single agent and in combination with TMZ or irinotecan, chemotherapies often used in relapsed patients. In an RD-ES (ATCC® HTB166™) Ewing's sarcoma (EWS-FLI1) s.c. xenograft model, treatment with single agent E7449 or TMZ resulted in no antitumor activity. However, when combined exquisite synergy that resulted in tumor regression and tumor-free mice was observed, even at the low E7449 combination dose of 1 mg/kg. E7449 dose responsive re-growth of tumors was observed and 8/8 mice remained tumor-free in the highest dose group (30 mg/kg) at study termination. Enhanced toxicity as measured by body weight loss was observed in combination treatment groups but mice generally recovered well. Significant antitumor activity was observed for irinotecan alone in the RD-ES model; combination with E7449 enhanced that activity. Combining TMZ and irinotecan was also efficacious in the RD-ES model. Antitumor activity was further enhanced by the addition of E7449 which also potentiated combination toxicity. Studies are ongoing to optimize dosing of E7449 and chemotherapy and to identify a treatment schedule that maximizes activity and tolerability of the various combinations. Additionally, the activity of E7449 alone and in combination was evaluated in 2 Ewing's sarcoma patient-derived xenograft (PDx) models. In one model (CTG-0143) neither TMZ nor E7449 as single agents had significant antitumor activity. However, the combination of E7449 + TMZ resulted in striking synergy and tumor regression. Although tumors were large when treatment started, significant antitumor activity was also observed with single agent irinotecan and combination treatment. The second model (CTG-0142) was sensitive to E7449 alone at high dose and to both chemotherapies (TMZ and irinotecan) as single agents. Addition of E7449 to either TMZ or irinotecan resulted in enhanced anti-tumor activity as measured by significantly delayed tumor re-growth following regression. Biomarker analysis is ongoing, including gene expression profiling and assessment of the DNA damage response in tumors from RD-ES and PDx models following drug treatments. In conclusion, the combination of E7449 with TMZ and irinotecan resulted in highly potent anti-cancer activity against cell line and PDx models of Ewing's sarcoma. These data support the assessment of E7449 as a combination therapy for Ewing's sarcoma in clinical trials. Citation Format: Sharon McGonigle, Zhihong Chen, Jingzang Tao Miu, Kuan-Chun Huang, Donna Kolber-Simonds, Nanding Zhao, Natalie C. Twine, Qiongfang Cao, Galina Kuznetsov, Shanqin Xu, Kenichi Nomoto. Antitumor activity of the PARP inhibitor E7449 in Ewing's sarcoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2733. doi:10.1158/1538-7445.AM2014-2733

8595 Background: E7080 is an oral TKI targeting VEGFR1-3, FGFR1-4, PDGFRβ, RET and KIT, which affects tumor cell proliferation and tumor vascularization in laboratory models. Tumor response (RECIST...

Recent studies strongly indicate that aberrations in the control of gene expression might contribute to the initiation and progression of Alzheimer's disease (AD). In particular, alternative splicing has been suggested to play a role in spontaneous cases of AD. Previous transcriptome profiling of AD models and patient samples using microarrays delivered conflicting results. This study provides, for the first time, transcriptomic analysis for distinct regions of the AD brain using RNA-Seq next-generation sequencing technology. Illumina RNA-Seq analysis was used to survey transcriptome profiles from total brain, frontal and temporal lobe of healthy and AD post-mortem tissue. We quantified gene expression levels, splicing isoforms and alternative transcript start sites. Gene Ontology term enrichment analysis revealed an overrepresentation of genes associated with a neuron's cytological structure and synapse function in AD brain samples. Analysis of the temporal lobe with the Cufflinks tool revealed that transcriptional isoforms of the apolipoprotein E gene, APOE-001, -002 and -005, are under the control of different promoters in normal and AD brain tissue. We also observed differing expression levels of APOE-001 and -002 splice variants in the AD temporal lobe. Our results indicate that alternative splicing and promoter usage of the APOE gene in AD brain tissue might reflect the progression of neurodegeneration.

MicroRNA’s (miR) are small noncoding RNA’s of 18–25 nucleotides that have a critical impact on gene regulation affecting cell growth & differentiation. Importantly, miR expression profiles can help distinguish normal cells from cancerous cells. This is particularly relevant to myelodysplastic syndromes both because of their heterogeneity as well as the difficulties associated with the early diagnosis of these disorders. We investigated the miR profiles of CD34+ and total nucleated cells (TNC) from different subtypes of MDS (n=135) and compared these to AML (n=20), normal CD34+ (n=5) and normal TNC’s (n=6). We used the Illumina universal array matrix to interrogate 470 validated miR’s from the Sanger mir-Base database and a further 265 putative miR’s from the literature. Array intensity data was analysed using the Partek Genomics Suite v6.3. Data was normalised using quantile normalisation and unchanging miR’s removed. A 1-way ANOVA was used to identify differentially expressed miR’s and a FDR correction applied to control for Type I errors. Our initial analysis compared TNC (n=6) with CD34 cells (n=5) from normal controls. As expected both groups clustered separately with mir-199a (p

The ‘5q- syndrome’ is characterized by macrocytic anaemia, normal to elevated platelet count, dyserythropoiesis, hypolobulated megakaryocytes and an interstitial deletion of the long arm of chromosome 5(q31–q32) as the sole karyotypic abnormality. In this study we have used the Affymetrix 250K high resolution SNP arrays to refine the 5q- syndrome commonly deleted region (CDR) and to explore undetected chromosomal aberrations in CD34+ cells and in constitutional tissue from 5q- syndrome patients at diagnosis (n=17) and following Lenalidomide treatment at haematological remission (n=9). To correlate genomic data with gene expression, genome-wide gene expression analysis on CD34+ cells was also performed using the Affymetrix U133plus2 arrays. We detected 35 chromosomal aberrations (3 deletions, 1 amplification and 31 regions of uniparental disomy (UPD) >2Mb) in 8 (35%) of patients unidentified by metaphase cytogenetic analysis. The median size of UPD was (4.03MB) (range: 2.49 – 30.31). Copy number changes identified were acquired aberrations, whereas UPD’s were also present in constitutional DNA. There were no common regions of overlap between any of the UPDs and no obvious change in gene expression correlating with the sites of UPD. SNP analysis precisely mapped the proximal and distal break points of the 5q deletion and clearly identified the proximal end of the common deleted region (CDR) to extend from 5q23.3 to the distal breakpoint 5q33.1. Our results did not identify any patients with isolated minimal deletions of 5q31 or the 5q32/33.1 regions as defined by Ebert et al, 2008. Furthermore, there were no regions of UPD identified on chromosome 5q in any of our patients. There were 3 genes located on the deleted 5q region that were >3 fold down-regulated; LOX (5q23–q31, Kif20a (5q31) and SLC22A4 (5q31.2). Gene set enrichment analysis (GSEA) of CD34 expression data from presentation samples identified the IL2 (p

Therapy-related myelodysplastic syndromes (t-MDS) and therapy-related acute myeloid leukaemia (ICD-O code 9920/3) (t-AML) represent a serious long term complication in patients successfully treated for primary malignancies (PM) using chemotherapy, radiation therapy or a combination of these therapies. Factors that contribute to the incidence of t-MDS/t-AML include, the type and duration of the therapeutic regime used to treat the PM and host predisposition. Conventional techniques to identify genomic aberrations in t-MDS/t-AML, suffer from low throughput and poor resolution. Recent developments in single-nucleotide polymorphisms (SNP) microarray technology have enabled the exploitation of SNP arrays for high-resolution genome-wide genotyping. We investigated the frequency of chromosomal aberrations and polymorphisms in the GSTM1, GSTT1, GSTP1 and NAT2 enzymes in patients developing t-MDS/t-AML following adjuvant therapy for breast cancer. Using 250K SNP arrays we have analysed two cohorts of similarly treated patients (median age 56 years, range 26–75 years), one group developing t-MDS/t-AML (+t-MDS) and the other remaining disease free (control) within the same follow up time. In all cases constitutive DNA was also simultaneously analysed. We detected multiple chromosomal aberrations including copy number aberrations (CNA) and UPD summarised in Table 1. Table 1 Amplifications Deletions UPD No.of pts (%) Median (range) Mb No. of pts (%) Median (range) Mb No. of pts (%) Median (range) Mb + t-MDS (n=17) 10 (48) 1.4(0.2–105) 13 (62) 8.8(0.04–125) 9 (43) 4.4(2.1–35.4) Control (n=30) 6 (20) 0.4(0.2–2.5) 9 (30) 0.3(0.2–0.7) 15 (50) 3(2–14) There is a distinct difference in CNA between both groups of patients. Interestingly, we identified CNA in the peripheral blood of patients (67%) not developing t-MDS/t- AML which was not observed in any of our normal control samples with no history of chemotherapy. The frequency of both deletions and amplifications was almost twice in + tMDS group in comparison to control samples. Furthermore, the frequency (43% vs. 50%) and median size of UPD (4.4Mb vs. 3Mb) was similar between both groups respectively. SNP array data was also used to test for an association with t-MDS/t-AML. Relative risk (RR) was estimated to determine the effect of each SNP on the risk of t-MDS/t-AML development, with patients not developing t-MDS/t-AML. SNP’s were included in the analysis if they physically mapped onto a gene, tagging SNP’s and met a significance criteria (p

The specificity of the clinicomorphological characteristics in 5q- syndrome has led to a search for putative tumour suppressor genes localised to the critical deleted region (CDR) of 5q-. To date, microarray expression studies in 5q- syndrome patients have been carried out on CD34+ cells, neutrophils or total bone marrow (BM) cells. We have used a Affymetrix microarray approach to investigate allelic imbalance (AI) and the transcription profile of CD61+ and CD34+ cells from patients (n=17) presenting with 5q- of which 10 have an “isolated 5q-“. the rest having additional cytogenetic abnormalities. BM CD34+ and CD61+ cells from normals (n=10) were controls in expression profiling. DNA or total RNA from CD61+ or CD34+ cells and constitutional DNA was prepared and hybridised to the 500K mapping or U113 plus 2.0 arrays. GC-RMA normalized expression data was analysed using non-parametric testing with Genespring GX software to generate lists of differentially regulated genes. DChip was used to analyse SNP data. From a list of changing genes 48/720 occur in both the CD61+ and CD34+ cell profiles. 16/48 of these genes map to 5q of which 10/16 map to 5q31. Of these genes KIF20A showed the most marked change (5 and 3.8 fold down) in CD34+ and CD61+ cells respectively. KIF20A is a microtubule-associated protein that is upregulated in mitosis and localizes to the spindle midzone during anaphase. Knock out of KIF20A protein results in binucleate nuclei indicating its involvement in both cytokinesis and nucleokinesis. Haploinsufficiency of this gene, may account for mononuclear megakaryocytes that are so characteristic of 5q- syndrome. Interestingly other members of the kinesin family expressed in 5q- CD34+ cells are down regulated in 5q- CD61+ cells (KIF21B, KIF2, KIF3A and KIF1B) indicating that in CD34+ cells, a compensatory mechanism for KIF20A haploinsufficiency may exist which is absent in megakaryocytes. The 5q- deletion (q14.2–q33.3) was detected in 6/10 CD61+ and 11/17 CD34+ cell populations by SNP analysis. The disparity reflects the admixture of normal and MDS CD61+ and CD34+ cells. Three patients presented with additional cytogenetic abnormalities, two had −7 or del 7(q22–q36), both being detected with the SNP array. The third patient had trisomy 8 that was not detected. SNP microarray identified 5q31.1–5q33.2 as the minimal deleted segment, which includes previously described CDR. Paired analysis of CD61+ and CD34+ with constitutional DNA revealed only 2/17 patients had additional abnormalities in CD34+ cells. One with a del12(q21.1–22) (21Mb). The 2nd had −13(q12.3–34;83Mb),+ 13(q11–12.3; 12.5Mb) and +11(q22.3-qterm; 30Mb) These changes were not detected by cytogenetic analysis. 2/6 patients shared 13Mb of UPD on chromosome 3(p12.2–q11.2) and chromosome 9(q33.1–q34.1) in CD34+ cells as well as constitutive DNA. We conclude that unlike AML, 5q- Syndrome does not have significant rate of UPD. Only a small number of genes are under expressed in both CD61+ and CD34+ cells and amongst these KIF20A is the most significant change and may be responsible for some of the clinicomorphological features of 5q- abnormality.

The myelodysplastic syndromes (MDS) encompass a heterogeneous group of clonal stem cell disorders characterised by ineffective erythropoiesis and peripheral blood cytopenias. However, specific subgroups of MDS with both myeloproliferative (MPD) and dysplastic features are recognized, which include chronic myelomonocytic leukaemia (CMML), refractory anaemia with ringed sideroblasts and thrombocytosis (RARS-T) and MDS/MPD unclassified. In addition cases of 5q- syndrome may present with a marked elevation in platelet count and a hypercellular bone marrow. The molecular events which determine the predominant morphological features however remain unclear. The JAK2 somatic point mutation (V617F) results in constitutive activation of the tyrosine kinase and culminates in both proliferation and differentiation signals to the cell and thus may contribute to the proliferative features identified in MDS as reported in the chronic MPDs. We analysed 194 patients from six centres with a diagnosis of MDS or MDS/MPD for the presence of the JAK2 mutation. 11 cases of RA; 70, RARS; 60, 5q- syndrome; 28, refractory anaemia with excess blasts (RAEB); 9, acute myeloid leukaemia with multi-lineage dysplasia (AML-MLD); 4, MDS/MPD-U; 1, CMML; and 1, hypoplastic MDS were analysed. DNA samples were obtained from bone marrow aspirate or peripheral blood. Ethical approval was obtained. Genomic DNA was prepared using standard methods (Qiagen). A modified allele specific PCR (AS PCR) was used to screen DNA samples. JAK2 mutated DNA samples were further subjected to pyrosequencing for confirmatory testing. The median age of the JAK2 mutant versus wild type (WT) cases was 66years (46–80years) v 67years (30–92years). The JAK2 mutation was detected in 13/194 (6.7%) cases overall. 4/70 (5.7%) RARS, 5/60 (8%) 5q- syndrome, 1/28 RAEB (associated with del 5q), 1/4 CMML, 1 MDS/MPD-U and 1/8 AML-MLD demonstrated the mutation. Within the RARS cohort, the prevalence of JAK2 mutation increased to 4/6 (67%) of cases with thrombocytosis (platelets >500x109/l). The presence of the mutation was confirmed in 8/13 cases. In cases analysed, the mutation was detected in CD34+, CD14+, CD15+ and CD61+ cell fractions but not CD3+ or CD19+ cell lineages. On analysis of the haemoglobin (Hb), platelet count and total white cell count (WCC) in the JAK2 mutant versus wild-type groups, there was no difference in median Hb (10.8 v 9.2 g/dl, p=0.275) but a significantly higher median platelet count (526 v 209 x109/l, p

2012 Background: Resistance to tubulin-targeted agents has been associated with differential tubulin isotype expression in cancer cells. In this study we investigated whether β-tubulin isotype comp...

Juvenile rheumatoid arthritis (JRA) comprises a group of chronic systemic inflammatory disorders that primarily affect joints and can cause long-term disability. JRA is likely to be a complex genetic trait, or a series of such traits, with both genetic and environmental factors contributing to the risk for developing the disease and to its progression. The HLA region on the short arm of chromosome 6 has been intensively evaluated for genetic contributors to JRA, and multiple associations, and more recently linkage, has been detected. Other genes involved in innate and acquired immunity also map to near the HLA cluster on 6p, and it is possible that variation within these genes also confers risk for developing JRA. We examined the TPSN gene, which encodes tapasin, an endoplasmic reticulum chaperone that is involved in antigen processing, to elucidate its involvement, if any, in JRA. We employed both a case–control approach and the transmission disequilibrium test, and found linkage and association between the TPSN allele (Arg260) and the systemic onset subtype of JRA. Two independent JRA cohorts were used, one recruited from the Rheumatology Clinic at Cincinnati Children's Hospital Medical Center (82 simplex families) and one collected by the British Paediatric Rheumatology Group in London, England (74 simplex families). The transmission disequilibrium test for these cohorts combined was statistically significant (χ2 = 4.2, one degree of freedom; P = 0.04). Linkage disequilibrium testing between the HLA alleles that are known to be associated with systemic onset JRA did not reveal linkage disequilibrium with the Arg260 allele, either in the Cincinnati systemic onset JRA cohort or in 113 Caucasian healthy individuals. These results suggest that there is a weak association between systemic onset JRA and the TPSN polymorphism, possibly due to linkage disequilibrium with an as yet unknown susceptibility allele in the centromeric part of chromosome 6.

CCI-779 (CCI) is a novel mTOR kinase inhibitor being developed for treatment of various cancers. In this randomized, double-blind, multicenter trial, 25, 75, or 250 mg IV CCI was given once weekly to 110 patients with renal cancer and was shown to be clinically active (Atkins et al. Proc ASCO 22:201,2003). Herein, the population pharmacokinetics (PPK) of CCI and sirolimus (SIR) were evaluated for dynamic relationship to safety, activity, and pharmacogenomic responses. With correlation analysis, we explored the relationship between gene expression changes in peripheral blood mononuclear cells (PBMC) and cumulative (∑)AUC and AUCsum (CCI+SIR) at 8 and 16 weeks of treatment. Exposures were related to adverse events (AE) and survival. Final PPK models of CCI and SIR included 235 and 305 observations, respectively, from 50 patients. CCI clearance = 1.39*(1+DNUM*0.103)*DOSE0.551 *BSA1.28 in which DNUM = 0/1 for single/multiple dose and BSA = body surface area. Age, sex, race, or renal risk factor did not affect disposition. AE severity correlated to AUCsum for thrombocytopenia, pruritis, and hyperlipemia (p < 0.05). Average AUC or AUCsum were not related to survival differences. At 16 weeks, individual ∑AUCsum was significantly correlated with change from baseline gene expression levels for 19 transcripts. Improved survival in the absence of an exposure relationship suggests that test doses are at or near a plateau of maximum response. A transcript subset from PBMCs significantly correlated with ∑AUCsum and may qualify as biomarkers of CCI exposure. Clinical Pharmacology & Therapeutics (2004) 75, P41–P41; doi: 10.1016/j.clpt.2003.11.155

BackgroundThe differentiation of human bone marrow derived skeletal stem cells (known as human bone marrow stromal or mesenchymal stem cells, hMSCs) into osteoblasts involves the activation of a small number of well-described transcription factors. To identify additional osteoblastic transcription factors, we studied gene expression of hMSCs during ex vivo osteoblast differentiation.ResultsClustering of gene expression, and literature investigation, revealed three transcription factors of interest – ZNF25, ZNF608 and ZBTB38. siRNA knockdown of ZNF25 resulted in significant suppression of alkaline phosphatase (ALP) activity. This effect was not present for ZNF608 and ZBTB38. To identify possible target genes of ZNF25, we analyzed gene expression following ZNF25 siRNA knockdown. This revealed a 23-fold upregulation of matrix metallopeptidase 1 and an 18-fold upregulation of leucine-rich repeat containing G protein-coupled receptor 5 and RAN-binding protein 3-like. We also observed enrichment in extracellular matrix organization, skeletal system development and regulation of ossification in the entire upregulated set of genes. Consistent with its function as a transcription factor during osteoblast differentiation of hMSC, we showed that the ZNF25 protein exhibits nuclear localization and is expressed in osteoblastic and osteocytic cells in vivo. ZNF25 is conserved in tetrapod vertebrates and contains a KRAB (Krueppel-associated box) transcriptional repressor domain.ConclusionsThis study shows that the uncharacterized transcription factor, ZNF25, is associated with differentiation of hMSC to osteoblasts.